Efficient identification of marker chromosomes in 27 patients by stepwise hybridization with alpha-satellite DNA probes

Using a procedure involving stepwise hybridization of alpha-satellite DNA probes at various conditions of stringency, 33 marker chromosomes from 27 patients were identified. The markers were ascertained prenatally in fetal amniotic fluid and chorionic villi samples or postnatally in blood from liveborn children. The marker chromosomes first were characterized by cytogenetic techniques and later identified by fluorescence in situ hybridization. There were 14 bisatellited markers, 3 metacentric nonsatellited marker chromosomes, 2 nonsupernumerary sex-chromosomal rings, and 9 patients carrying markers that appeared to be small rings. Multiple stringency conditions were used for the identification of 14 supernumerary ringlike chromosomes detected in 8 patients. Ring-like markers were initially screened at low stringency and grouped into alpha-satellite families. Subsequent higher stringency hybridization led to marker identification. Ringlike chromosomes originated from chromosomes 1, 2, 8, 12, 13 or 21, 14 or 22, 15, 18, and X. Multiple ringlike markers ascertained in a single patient were determined to originate from different chromosomes.     

Multiplex-FISH for pre- and postnatal diagnostic applications.

For >3 decades, Giemsa banding of metaphase chromosomes has been the standard karyotypic analysis for pre- and postnatal diagnostic applications. However, marker chromosomes or structural abnormalities are often encountered that cannot be deciphered by G-banding alone. Here we describe the use of multiplex-FISH (M-FISH), which allows the visualization of the 22 human autosomes and the 2 sex chromosomes, in 24 different colors. By M-FISH, the euchromatin in marker chromosomes could be readily identified. In cases of structural abnormalities, M-FISH identified translocations and insertions or demonstrated that the rearranged chromosome did not contain DNA material from another chromosome. In these cases, deleted or duplicated regions were discerned either by chromosome-specific multicolor bar codes or by comparative genomic hybridization. In addition, M-FISH was able to identify cryptic abnormalities in patients with a normal G-karyotype. In summary, M-FISH is a reliable tool for diagnostic applications, and results can be obtained in 相似文献   

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro

Summary Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome number between 49 and 52. Analysis of Wright’s-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10. This work was supported by grants-in-aid made available through the San Diego State University Foundation.     

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.     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro.

Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome mumber between 49 and 52. Analysis of Wright's-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10.     

Inheritance of acrocentric association patterns.

The individual association frequencies of acrocentric chromosomes identified by fluorescent markers were analyzed in 17 individuals from 3 families. The frequency of association appears to be a characteristic property of an individual chromosome, since certain marker chromosomes showed an increase in frequency of association in each family member in which they appeared. Most of the marker chromosomes with increased frequency of association had a longer nucleolar constriction than their homologs.     

Tetrasomy 15q: two marker chromosomes with no detectable alpha-satellite DNA.

Two patients with specific and similar phenotypes were both found to have an unusual marker chromosome present in 70%-80% of their lymphocytes at routine cytogenetic examination. The marker chromosomes were isolated by flow sorting and were amplified by degenerate oligonucleotide-primed PCR. These libraries and a cosmid probe located at 15q26 were used to characterize the marker chromosomes by FISH. Both marker chromosomes were found to consist of duplicated chromosome material from the distal part of chromosome 15q and were identified as inv dup(15) (qter-->q23::q23-->qter) and inv dup(15) (qter-->q24::q24-->qter), respectively. Hence, the markers did not include any known centromere region, and no alpha-satellite DNA could be detected at the site of the primary constriction. Tetrasomy 15q may be a new syndrome, associated with a specific type of marker chromosome. In addition, further analyses of this type of marker chromosome might give new insight into the structure and function of the mammalian centromere.     

Analysis of replication patterns in chromosomes of normal Chinese hamster and its cell lines

Replication patterns of the normal male Chinese hamster chromosomes and the three cell lines CHW, 1102 and 1103, were determined using fluorescent, plus Giemsa or acridine orange, techniques. The individual chromosomes or chromosomal segments were consistent in the replication patterns of normal Chinese hamster chromosomes and all the transformed cell lines. Late DNA replication was regularly identified in the long arm of the X chromosome, the entire Y chromosome, the short arms of chromosomes 6 and 7, and the paracentromeric regions of chromosomes 8, 9 and 10. A similar consistency was demonstrated in the large late replicating areas of chromosomes X and Y. Each cell line had specific marker chromosomes by which the cell line was identified and their replication patterns have been described. The chromosome analysis in cell line 1103 indicated that chromosomes 2, 3, 8 and 9 were more stable than others, of which chromosome 2 was extremely stable. The markers M4 and M5 in cell line 1103 are very interesting. The cytogenetic behaviour of marker M4 indicated a new phenomenon of translocation by simple association. The marker chromosome M5 indicated that inactivation spread to the early replicating distal region. These cell lines are very useful tools for studying replication patterns and providing a basic understanding of mammalian cytogenetics.     

Identification and characterization of normal length nonfluorescent Y chromosomes: cytogenetic analysis,Southern hybridization and non-isotopic in situ hybridization

Summary In two female patients with a 45,X/46,X,+mar karyotype the marker chromosomes were identified as normal length nonfluorescent Y chromosomes (nlY^nf) using non-isotopic in situ hybridization (NISH) complementary to routine cytogenetic analysis and Southern hybridization. The recognition of the nlY^nf as isodicentric in both patients illustrates and confirms the usefulness and importance of NISH in the identification and characterization of this and many other types of complex chromosome rearrangements.     

Molecular characterization of the marker chromosome associated with cat eye syndrome.

Cat eye syndrome (CES) is associated with a supernumerary bisatellited marker chromosome which is derived from duplicated regions of 22pter-22q11.2. In this study we have used dosage and RFLP analyses on 10 CES patients with marker chromosomes, by using probes to five loci mapped to 22q11.2. The sequences recognized by the probes D22S9, D22S43, and D22S57 are in four copies in all patients, but the sequences at the more distal loci, D22S36 and D22S75, are duplicated only in some individuals. D22S36 is present in three copies in some individuals, and D22S75 is present in two copies in the majority of cases. Only three individuals have a duplication of the most distal locus examined (D22S75), and these individuals have the largest marker chromosomes identified in this study. From the dosage analysis it was found that the marker chromosomes are variable in size and can be asymmetric in nature. There is no obvious correlation between the severity of the phenotype and the size of the duplication. The distal boundary of the CES critical region (D22S36) is proximal to that of DiGeorge syndrome, a contiguous-gene-deletion syndrome of 22q11.2.     

Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization.

The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.     

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.     

Characterization of human heteroploid cell line J-111: Reverse banding patterns of marker chromosomes

The karyotype of the human cell line, J-111, has been studied employing R-banding by fluorescence using acridine orange technique (RFA). The model chromosome number of this line was 112. All human chromosomes except the Y were present in each metaphase. Twenty-one marker chromosomes were distinguished and their possible origins were investigated. Of these, twelve were consistently present in all cells. Nine markers were highly variable. Four typical marker chromosomes of HeLa cells were found and their origins were identified, indicating that the line is a HeLa contaminant. The reverse banding patterns of all marker chromosomes are presented and the value of the RFA technique is discussed.     

Mosaicism in 45,X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes?

Summary Cytogenetic and molecular genetic findings in 91 patients with Turner syndrome are reported. In 87 patients, chromosome studies were carried out both in lymphocyte and fibroblast cultures. Mosaicism was demonstrated in 58 of these patients (66.7%), whereas only 18 (20.7%) were apparent non-mosaic 45,X, and 11 patients (12.6%) showed non-mosaic structural aberrations of the X chromosome. Among the mosaic cases 16 (18.4% of all patients) displayed a second cell line containing small marker chromosomes. The association of Y-specific chromosomal material with the presence of marker chromosomes was demonstrated in 6 out of 7 mixoploid fibroblast cell lines by polymerase chain reaction amplification and by Southern-blot analysis. The observation of ring formation and morphological variability in vivo and in vitro, and the continous reduction in the percentage of cells containing marker chromosomes in longterm cultivation experiments indicated an increased instability of marker chromosomes. The findings suggest that in vivo selection of structurally altered sex chromosomes exists. Thus, the observation of apparent non-mosaic 45,X chromosomal complements in liveborn individuals with Turner syndrome does not contradict the hypothesis that some degree of mosaicism is necessary for survival in early pregnancy.     

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.     

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.     

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.     

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.     

Characterization of seven DA/DAPI-positive bisatellited marker chromosomes by in situ hybridization

Summary Seven dicentric bisatellited marker chromosomes, ascertained at amniocentesis, chorionic villus sampling, and in blood from an abnormal liveborn were characterized cytogenetically. All seven markers demonstrated brilliant bands by the DA/DAPI technique corresponding to C-band positive regions. Although some dicentric DA/DAPI-positive bisatellited markers have been identified as inverted duplicated 15s, recent literature has suggested that DA/DAPI lacks specificity for chromosome 15. Our evaluation of DA/DAPI-positive bisatellited marker chromosomes by in situ hybridization shows that some originate from chromosome 15 whereas DA/DAPI negative bisatellited markers may not be derived from 15. The morphological variations noted in our studies are discussed with respect to nomenclature.     

Two supernumerary marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism

The interpretation of the significance of marker chromosomes, which can be encountered at prenatal diagnosis, is extremely problematic. Various factors contribute to the difficulty of clarifying the phenotypic risks of supernumerary marker chromosomes, including differences in the size, structure, and origin of marker chromosomes, as well as the occurrence of multiple marker chromosomes of different origin in the same proband. Research on marker chromosomes is currently in a data-accumulation phase. We report the presence of two marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism and discuss the related literature.