Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].     

Complete and precise characterization of marker chromosomes by application of microdissection in prenatal diagnosis

A straightforward and extremely efficient reverse chromosome painting technique is described which allows the rapid and unequivocal identification of any cytogenetically unclassifiable chromosome rearrangement. This procedure is used to determine the origin of unknown marker chromosomes found at prenatal diagnosis. After microdissection of the marker chromosome and amplification of the dissected fragment by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), fluorescence in situ hybridization (FISH) to aberrant and normal metaphase chromosomes with the marker-derived probe pool is performed. With this strategy, marker chromosomes present in amniotic fluid samples were successfully identified in three cases. The origin of the supernumerary markers was ascertained as deriving from 3p(p12-cen), 18p(pter-cen) and 9p(p12-cen), respectively. Since a specific FISH signal on chromosomes can be obtained within 2 working days using a probe generated without any pretreatment from one chromosomal fragment only and without additional image processing devices, this technique is considered to be highly suitable for routine application in pre- and postnatal cytogenetic analysis.     

The significance of accessory bisatellited marker chromosomes in amniotic fluid cell cultures

Thirteen new cases of accessory bisatellited marker chromosomes were found among 20,370 amniocentesis. Six of these were familial, six originated de novo and in one case the origin of marker chromosome remained unknown. Eleven cases were carried to term and follow up studies revealed no abnormality. In two cases the pregnancies were terminated and the pathological examination revealed apparently normal fetuses. A cytogenetic categorization of bisatellited marker chromosomes is described. The available data show clearly that there is no increased risk for offspring with abnormal phenotype born to a healthy carrier of an accessory bisatellited marker chromosome with either a single or two closely adjacent C-bands (category AI or AII). The unbiased sample of cases with de novo accessory bisatellited marker chromosomes of category AI or AII is still too small to allow a satisfactory estimation. However, the actual risk for a fetus to be affected may be low too.     

Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X–linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.     

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients

Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients' genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1-->q12:) accompanied by a sSMC(18): r(18)(:p11.2-->q11.1::p11.2-->q11.1:), inv dup(18)(:p11.1-->q11.1::q11.1-->p11.1:), or der(18) (:p11.2-->q11.1::q11.1-->p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12-->q11.1::q11.1-->p21:) der(8) (:p11.22-->q11.1::q11.1-->p21::p21-->p11.22:) and der(21)(:p11.1-->q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes.     

The genetic significance of accessory bisatellited marker chromosomes

Ten new cases of accessory bisatellited marker chromosomes examined in different laboratories are reported. As a basis for genetic counseling in the context of prenatal diagnosis a cytogenetic categorization of such marker chromosomes is proposed and an estimation of the genetic risk associated with each category is carried out. The results are as follows: There is no increased risk for offspring with abnormal phenotype born to a healthy carrier of an accessory bisatellited marker chromosome with either a single or two closely adjacent C-bands (Category AI or AII). The unbiased sample of cases with de novo accessory bisatellited marker chromosomes of categories AI and AII is too small to allow a satisfactory estimation of the actual risk that, in case of such a prenatal finding, the foetus may not show a normal phenotype as a consequence of the marker chromosome. There is, however, evidence that this risk may be lower than 10%. Accessory bisatellited marker chromosomes showing a discrete pattern of G- and R-bands situated between two distant C-bands (Category AIII) usually indicate a chromosomal imbalance giving rise to an abnormal phenotype. Mosaic carriers of such dicentric marker chromosomes may, however, present a normal phenotype.     

Chromosomal origin of small ring marker chromosomes in man: characterization by molecular genetics.

Ten cases of small ring chromosomes which did not stain with distamycinA/DAPI and did not possess satellite regions associated with nucleolus-organizing regions are described. In situ hybridization with a battery of biotinylated pericentric repeat probes specific either for individual chromosomes or for groups of chromosomes allowed the identification of the chromosomal origin of these marker chromosomes. There was one example of a marker derived from each of chromosomes 1, 3, 6, 14, 16, 18, 20, 13 or 21, and the X, and there were two examples of markers derived from chromosome 12. One case possessed two markers, one derived from chromosome 6, and one derived from the X. The mechanism of generation of ring marker chromosomes is discussed. Five of seven cases who could be phenotypically assessed were abnormal. Three of these--the first with a ring chromosome derived from chromosome 1; the second with two markers, one derived from chromosome 6 and the other from the X chromosome; and the third with a ring chromosome derived from chromosome 20--each possessed distinctive facies. Additional cases with identified rings may allow the delineation of new chromosomal syndromes.     

Autosomal dominant retinitis pigmentosa: exclusion of a gene from extensive regions of chromosomes 6, 13, 20, and 21

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for polymorphic DNA markers from chromosomes 6, 13, 20, and 21. For each marker close linkage to ADRP has been excluded by pairwise analyses. Using distances fixed from well-established genetic maps of these chromosomes and multipoint analyses with two or three contiguous markers, exclusion of ADRP was extended to the areas between markers, resulting in the exclusion of ADRP from extensive regions of each chromosome, totaling approximately 500 cM or 15% of the genome. The study indicates the large quantity of linkage/exclusion data obtainable using well-spaced highly polymorphic markers.     

Ors12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.     

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae)

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlantica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.     

Marker chromosomes commonly observed in the genus Glycine

Summary Soybean [Glycine max (L.) Merr.] chromosomes were analyzed using the chromosome image analyzing system, CHIAS, and seven groups, including subgroups, were identified based on morphological characteristics. Two pairs of chromosomes were conspicuous in their morphological traits. One pair of chromosomes, which had the largest arm ratio among all the chromosomes, was commonly observed in the species in all three subgenera of the genus Glycine. These chromosomes also displayed a unique pattern after N-banding and were detected as marker chromosomes. G. soja, which is considered to be the ancestor of G. max, has two types of marker chromosomes. The lines that carry the same type as G. max may be the ancestors of G. max among the lines of G. soja. The morphological differences of the marker chromosomes within the species in the subgenus Soja are discussed in relation to the domestication process of soybean.     

Marker chromosomes of murine cell lines

Using the C-method of chromosome staining four marker chromosomes were revealed in the transplanted murine line SC-1, one comparatively rare marker chromosome was shown in RAG line, small marker chromosomes occurred almost in all cells of RVP3 line. Marker chromosomes found in the studied lines by the C-method of chromosome staining make it possible to distinguish these lines from each other.     

De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints.

A questionnaire sent to major cytogenetics laboratories in the United States and Canada over a 10-year period collected data on the frequency and outcome of cases with either apparently balanced de novo rearrangements or de novo supernumerary marker chromosomes detected at amniocentesis. Of 377,357 reported amniocenteses, approximately 1/2,000 had a de novo reciprocal translocation, 1/9,000 a Robertsonian translocation, 1/10,000 a de novo inversion, and 1/2,500 an extra structurally abnormal chromosome of unidentifiable origin. The risk of a serious congenital anomaly was estimated to be 6.1% (n = 163) for de novo reciprocal translocations, 3.7% (n = 51) for Robertsonian translocations, and 9.4% (n = 32) for inversions. The combined risk for reciprocal translocations and inversions was 6.7% (95% confidence limits 3.1%-10.3%). The risk of abnormality for extra nonsatellited marker chromosomes was 14.7% (n = 68), and that for satellited marker chromosomes was 10.9% (n = 55). In non-Robertsonian rearrangements, distribution of breakpoints among chromosomes was not as would be expected strictly on the basis of length. Most breaks were stated to occur within G-negative bands, but there was little evidence of particular hot spots among these bands. Nevertheless, there did appear to be a correlation between those bands in which breakage was observed most often and those bands where common or rare fragile sites have been described.     

Incidence and significance of supernumerary marker chromosomes in prenatal diagnosis

The finding of a supernumerary marker chromosome in amniotic fluid cells poses a considerable counseling dilemma. In 6,500 cases referred to our laboratory over a 4 1/2-year period, eight such cases were identified (0.123% of all cases). In five of the eight cases, a diagnosis of true mosaicism between cells with 46 and 47 chromosomes was made. In the remaining three cases, the marker was present in 100% of the cells. In three cases, the marker was determined to be familial in nature with mosaicism present in the parents of two of these cases. Detailed cytogenetic findings for each case are provided. In no cases were abnormalities noted in either abortuses or live borns. The high incidence of mosaicism in these cases seems to indicate a propensity for supernumerary chromosomes to be lost. Familial markers may not be passed on for many generations, and they may arise as new mutations relatively frequently. There is an urgent need for more information on the risks associated with the prenatal detection of supernumerary chromosomes. We recommend that in considering the implications of the prenatal detection of marker chromosomes cases be considered in at least four distinct groups: type 1--familial and nonmosaic; type 2--familial with mosaicism in either the amniotic fluid cells, a parent, or both; type 3--de novo markers and nonmosaic; and type 4--de novo with mosaicism present in the amniotic fluid cells.     

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding,CGH, M-FISH,and chromosome- and locus-specific FISH

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.     

Centromere organization in chromosomes of the mouse

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.     

De novo origin of multiple small supernumerary marker chromosomes (sSMCs) in a child with intellectual disability and dysmorphic features

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.     

Heterochromatin heterogeneity and rapid divergence of the sex chromosomes in Chorthippus parallelus parallelus and C. p. erythropus (Orthoptera).

Mitotic chromosomes from individuals of a Pyrenean hybrid zone between two subspecies of the grasshopper Chorthippus parallelus have been pretreated as for C-banding and subsequently stained with 4,6-diamidino-2-phenylindole (DAPI) and (or) Chromomycin A3 (CMA). The results, which are different from those obtained with Giemsa C-banding or standard DAPI - CMA treatments show (i) hidden heterochromatic heterogeneity that may be correlated with the existence of distinct families of repetitive DNAs, (ii) information about the possible independent origin of the three detected types of heterochromatin, and (iii) a further marker difference between the sex chromosomes of these two subspecies. This last result leads us to discuss the possible differential rates of evolution of sex chromosomes and autosomes in these subspecies and provides us with a new tool for the study of the structure and dynamics of this hybrid zone.     

An improved technique for sequential R-, Q- and C-banding of bone marrow chromosomes

A procedure for the sequential staining of metaphase cells to provide R-banding, Q-banding and C-banding of chromosomes is described. The combined techniques allow more precise identification of marker chromosomes and more accurate definition of breakpoints in the consistent chromosome translocations that are associated with certain human malignancies.