Prediction of clonal chromosome aberration frequency in human blood lymphocytes

We recently conducted a large-scale screening for clonal aberrations among atomic bomb survivors and proposed a model for the gross clonal composition of blood lymphocytes. Here we show an application of the model indicating that the number, m,of clones detectable by cytogenetic methods in an individual is predictable by the equation m= (1.8 + 6.4FG) x FP x n/500, where FG represents the estimated translocation frequency in the 46 chromosome set, FP is the observed translocation frequency with FISH or other methods, and nis the number of cells examined. Application of the equation to the results of seven other reports gave close agreement between the observed and calculated numbers of clones. Since the model assumes that clonal expansion is ubiquitous, and any translocation can be the constituent of a clone detectable by cytogenetic means, the vast majority of observed clonal expansions of these somatic cells are likely the result of random-hit events that are not detrimental to human health. Furthermore, since our model can predict the majority of clonal aberrations among Chernobyl workers who were examined 5-6 years after irradiation, clonal expansion seems to occur primarily within a few years after exposure to radiation, most likely being coupled with the process of recovery from radiation-induced injury in the lymphoid and hematopoietic systems.     

Differential cell division in human X chromosome mosaics

Summary Fibroblasts were grown from skin explants of 3 human females who are sex chromosome mosaics. The 3 cultures had the following chromosome complements: 45,X/46,XX, 45,X/46,XXqi and 45,X/47,XXX. Using thymidine labelling and Colcemid accumulation of metaphases it was found that in all 3 mosaic cultures the 45,X cells had a faster cell cycle than the second cell population. The difference in cell cycle duration was attributed to the longer G₁ phase in the cells with 2 or 3 X chromosomes. The 2 populations of cells in the mosaic only differ in the number of heterochromatic X chromosomes and it is argued that heterochromatin has a retardative effect on cell division.

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Zusammenfassung Kulturen von Fibroblasten der Haut dreier weiblicher Individuen mit Sexchromatin-Mosaiks zeigten folgende chromosomale Zusammensetzung: 45,X/46,XX; 45,X/46XXqi und 45,X/47,XXX. Mit Hilfe von Thymidinmarkierung und Akkumulation von Metaphasen durch Colcemid konnte gezeigt werden, daß in allen 3 Mosaikkulturen die 45,X-Zellen einen schnelleren Zellcyclus haben als die zweite Zellpopulation. Der Unterschied in der Dauer des Zellcyclus beruht auf der längeren G₁-Phase der Zellen mit 2 oder 3 X-Chromosomen. Die 2 Zellpopulationen im Mosaik differieren nur in der Zahl der heterochromatischen X-Chromosomen, so daß angenommen werden könnte, daß Heterochromatin einen retardierenden Effekt ausübt.

     

Inactivation centers in the human X chromosome.

Reported cases with a structurally abnormal X chromosome were compiled. These included 17 balanced and 26 unbalanced X-autosome translocations, each with inactivation of either a derivative X or a derivative of any of the autosomes. A further 52 cases with various structural rearrangements were studied. The shortest late-replicating segment in each arm pter leads to p21 and q13 leads to qter. In both cases, they were detected in all or most metaphases, thus making the results convincing. In one case, the distal part of Xq, q25 or 26 leads to qter was probably inactivated in a small proportion of the cells. It appears reasonable to assume that the former two segments and probably also the third include an "inactivation center(s)." In a male with a 46,Y,dup(X)(q13q22), no part of dup X replicated late although it contained extra chromosome material.     

X chromosome constitution and the human female phenotype

Summary The correlations of abnormal X chromosome constitutions and the resulting phenotypes in the human female are reviewed. The following hypotheses put forward to explain these correlations are discussed in detail: (1) The damage is done before X inactivation; (2) An effect is exerted between reactivation of the X chromosome(s) and meiosis in oocytes; (3) A recessive gene(s) in hemizygous condition might be expressed in the cases in which the same X is active in all cells; (4) A change in the number of presumed active regions on the inactive X chromosomes might have an effect; (5) A position effect, in that the region Xq13-q27 has to be intact in both X chromosomes to allow normal development, may be responsible; (6) An effect during the period when cells with different inactivation patterns compete is a probability; (7) The original X inactivation may be neither regular nor random.The conclusion reached is that the phenotypic effects of a specific X chromosome aberration may be simultaneously exerted through different pathways (Tables 1 and 2). Hypotheses (2), (4), (5), and (6) are considered probable. Hypothesis (3) has been discarded, and there is very little evidence for hypotheses (1) and (7).     

X chromosome inactivation in human and mouse pluripotent stem cells

Since the groundbreaking hypothesis of X chromosome inactivation (XCI) proposed by Mary Lyon over 50 years ago, a great amount of knowledge has been gained regarding this essential dosage compensation mechanism in female cells. For the mammalian system, most of the mechanistic studies of XCI have so far been investigated in the mouse model system, but recently, a number of interesting XCI studies have been extended to human pluripotent stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Emerging data indicate that XCI in hESCs and hiPSCs is much more complicated than that of their mouse counterparts. XCI in human pluripotent stem cells is not as stable and is subject to environmental influences and epigenetic regulation in vitro. This mini-review highlights the key differences in XCI between mouse and human stem cells with a greater emphasis placed on the understanding of the epigenetic regulation of XCI in human stem cells.     

Accelerated telomere shortening in the human inactive X chromosome

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.     

Large-scale mapping and chromosome jumping in the q27 region of the human X chromosome

We have used pulsed field gel electrophoresis for further physical mapping studies in the q27 region of the human X chromosome. We show that the DXS 102 locus and the F9 gene are separated by only 300 kb despite a genetic distance of 1.4 cM; this linkage orients our large-scale map and shows that the mcf.2 transforming sequence is telomeric to F9. A BssHII complete-digest jumping library was used to jump toward the DXS 105 locus; a 130-kb jump was achieved and the corresponding "linking clone" was obtained.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

Replication variants of the human inactive X chromosome

The sequence of DNA replication was studied within the inactive X chromosome in human lymphocytes, by means of the FPG method. Several variants of the replication sequence were found. The number of variants in the cells of a single donor exceeded 2 in each of the 4 normal individuals studied. The phenomenon is discussed with respect to the regulation of DNA synthesis and to the cell differentiation process.     

Evolution of pericentromeric heterochromatin of human X chromosome

An unusual large heterochromatic segment around the pericentromeric region of the X-chromosome is reported. In normal circumstances, the pericentromeric region of the X-chromosome is negative by the restriction endonuclease AluI/Giemsa technique. However, this unusual X-chromosome was found to have AluI resistant (positive) chromatin. The evolution of extra heterochromatin is a postzygotic event as substantiated by the presence of a normal cell line.     

Fragile X chromosome and chromosome condensation

The effect of chromosome condensation on the frequency of expression of the fragile X chromosome was examined. Chromosome decondensation substances were tested for their ability to elicit expression or improve frequencies of expression of the fragile X chromosome in five patients. The substances tested included the AT specific DNA ligands ethidium bromide, Hoechst 33258, and netropsin, and the GC specific substances actinomycin D and olivomycin. Under culture conditions appropriate for eliciting fragile X expression none of the decondensation compounds studied significantly altered frequencies of expression, nor did any of the substances elicit fragile X expression under conditions that normally suppress fragile X expression. The fragile X was found to be more frequently evident in less condensed chromosome preparations from fibroblasts. The implications of these findings with respect to the nature of fragile sites are discussed.     

Targeted sequencing of the human X chromosome exome

We used a RainDance Technologies (RDT) expanded content library to enrich the human X chromosome exome (2.5 Mb) from 26 male samples followed by Illumina sequencing. Our multiplex primer library covered 98.05% of the human X chromosome exome in a single tube with 11,845 different PCR amplicons. Illumina sequencing of 24 male samples showed coverage for 97% of the targeted sequences. Sequence from 2 HapMap samples confirmed missing data rates of 2–3% at sites successfully typed by the HapMap project, with an accuracy of at least ~ 99.5% as compared to reported HapMap genotypes. Our demonstration that a RDT expanded content library can efficiently enrich and enable the routine sequencing of the human X chromosome exome suggests a wide variety of potential research and clinical applications for this platform.     

Activation status of the X chromosome in human micronucleated lymphocytes

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon. Received: 5 May 1995 / Revised: 15 July 1995     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.