Identification of the translocation breakpoints in the Ts65Dn and Ts1Cje mouse lines: relevance for modeling down syndrome

Down syndrome (DS) is the most frequent genetic disorder leading to intellectual disabilities and is caused by three copies of human chromosome 21. Mouse models are widely used to better understand the physiopathology in DS or to test new therapeutic approaches. The older and the most widely used mouse models are the trisomic Ts65Dn and the Ts1Cje mice. They display deficits similar to those observed in DS people, such as those in behavior and cognition or in neuronal abnormalities. The Ts65Dn model is currently used for further therapeutic assessment of candidate drugs. In both models, the trisomy was induced by reciprocal chromosomal translocations that were not further characterized. Using a comparative genomic approach, we have been able to locate precisely the translocation breakpoint in these two models and we took advantage of this finding to derive a new and more efficient Ts65Dn genotyping strategy. Furthermore, we found that the translocations introduce additional aneuploidy in both models, with a monosomy of seven genes in the most telomeric part of mouse chromosome 12 in the Ts1Cje and a trisomy of 60 centromeric genes on mouse chromosome 17 in the Ts65Dn. Finally, we report here the overexpression of the newly found aneuploid genes in the Ts65Dn heart and we discuss their potential impact on the validity of the DS model.     

Early Lineage Priming by Trisomy of Erg Leads to Myeloproliferation in a Down Syndrome Model

Down syndrome (DS), with trisomy of chromosome 21 (HSA21), is the commonest human aneuploidy. Pre-leukemic myeloproliferative changes in DS foetal livers precede the acquisition of GATA1 mutations, transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL). Trisomy of the Erg gene is required for myeloproliferation in the Ts(17¹⁶)65Dn DS mouse model. We demonstrate here that genetic changes specifically attributable to trisomy of Erg lead to lineage priming of primitive and early multipotential progenitor cells in Ts(17¹⁶)65Dn mice, excess megakaryocyte-erythroid progenitors, and malignant myeloproliferation. Gene expression changes dependent on trisomy of Erg in Ts(17¹⁶)65Dn multilineage progenitor cells were correlated with those associated with trisomy of HSA21 in human DS hematopoietic stem and primitive progenitor cells. These data suggest a role for ERG as a regulator of hematopoietic lineage potential, and that trisomy of ERG in the context of DS foetal liver hemopoiesis drives the pre-leukemic changes that predispose to subsequent DS-TMD and DS-AMKL.     

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion. Received: 28 July 1998 / Accepted: 15 December 1998     

Trisomic and allelic differences influence phenotypic variability during development of Down syndrome mice

Individuals with full or partial Trisomy 21 (Ts21) present with clinical features collectively referred to as Down syndrome (DS), although DS phenotypes vary in incidence and severity between individuals. Differing genetic and phenotypic content in individuals with DS as well as mouse models of DS facilitate the understanding of the correlation between specific genes and phenotypes associated with Ts21. The Ts1Rhr mouse model is trisomic for 33 genes (the "Down syndrome critical region" or DSCR) hypothesized to be responsible for many clinical DS features, including craniofacial dysmorphology with a small mandible. Experiments with Ts1Rhr mice showed that the DSCR was not sufficient to cause all DS phenotypes by identifying uncharacteristic craniofacial abnormalities not found in individuals with DS or other DS mouse models. We hypothesized that the origins of the larger, dysmorphic mandible observed in adult Ts1Rhr mice develop from larger embryonic craniofacial precursors. Because of phenotypic variability seen in subsequent studies with Ts1Rhr mice, we also hypothesized that genetic background differences would alter Ts1Rhr developmental phenotypes. Using Ts1Rhr offspring from two genetic backgrounds, we found differences in mandibular precursor volume as well as total embryonic volume and postnatal body size of Ts1Rhr and nontrisomic littermates. Additionally, we observed increased relative expression of Dyrk1a and differential expression of Ets2 on the basis of the genetic background in the Ts1Rhr mandibular precursor. Our results suggest that trisomic gene content and allelic differences in trisomic or nontrisomic genes influence variability in gene expression and developmental phenotypes associated with DS.     

In vitro induction of numerical changes in the karyotype of normal and transformed Djungarian and Chinese hamster cells]

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.     

A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.     

The “happy puppet” syndrome in two siblings

Summary Disomic and trisomic cells of a patient with Down syndrome mosaic were used to study the effect of the additional chromosome 21 against an identical genetic background. The frequency of Ag staining and the participation in satellite associations were determined for each pair of acrocentric chromosomes. The additional chromosome 21 of the trisomic cells and its homologues proved to be regularly Ag positive. Therefore the trisomic cells showed more Ag positive chromosomes and more satellite associations per cell than the diploid cells. Thus, no compensation for the additional rRNA-gene dose could be found in the cells of the trisomic line.     

A FINE STRUCTURAL STUDY OF THE HIPPOCAMPUS AND DORSAL ROOT GANGLION IN MOUSE TRISOMY 16, A MODEL OF DOWN'S SYNDROME

Mouse trisomy 16 (Ts16) appears to provide an animal model of Down's syndrome in that a portion of mouse chromosome 16 is syntenic with part of human chromosome 21. Trisomy 21 in human beings leads to the mental retardation of Down's syndrome and in middle age, to some presenile anatomic and clinical features of Alzheimer's disease. Neural tissue from aging Ts16 mice is unavailable, however, as Ts16 mouse embryos die late in utero. We studied these embryos looking at the ultrastructure of neurons from the hippocampus and dorsal root ganglion in normal control mice embryos (diploid) and in Ts16 late embryonic litter mates after day 15 of gestation. The organelles in the Ts16 neurons looked similar to those in control neurons, fixed and processed under similar conditions. No obvious neuropathological structures were observed. These results, when compared to reports on electrophysiological abnormalities of cultured fetal Ts16 neurons and on abnormalities in neurotransmitter markers in the Ts16 fetal brain, lead us to suggest that the mental retardation of Down's syndrome is likely to result from functional and chemical defects not directly related to abnormal neuronal ultrastructure. When related to fine structural studies of transplanted embryonic Ts16 hippocampus which have been maintained for long periods of time, these results indicate that the trisomic mouse brain would not be useful as a structural model for Down's syndrome and hence presenile Alzheimer's disease, as it is not associated with any detectable morphological abnormality.     

Inducing segmental aneuploid mosaicism in the mouse through targeted asymmetric sister chromatid event of recombination

Loss or gain of whole chromosomes, or parts of chromosomes, is found in various pathological conditions, such as cancer and aneuploidy, and results from the missegregation of chromosomes during cellular division or abnormal mitotic recombination. We introduce a novel strategy for determining the consequences of segmental aneuploid mosaicism, called targeted asymmetric sister chromatin event of recombination (TASCER). We took advantage of the Cre/loxP system, used extensively in embryonic stem cells for generating deletions and duplications of regions of interest, to induce recombination during the G2 phase. Using two loxP sites in a Cis configuration, we generated in vivo cells harboring microdeletions and microduplications for regions of interest covering up to 2.2 Mb. Using this approach in the mouse provides insight into the consequences of segmental aneuploidy for homologous regions of the human chromosome 21 on cell survival. Furthermore, TASCER shows that Cre-induced recombination is more efficient after DNA replication in vivo and provides an opportunity to evaluate, through genetic mosaics, the outcome of copy number variation and segmental aneuploidy in the mouse.     

Mouse trisomy 16 as an animal model of human trisomy 21 (Down syndrome): production of viable trisomy 16 diploid mouse chimeras

We have previously proposed that mice trisomic for chromosome 16 will provide an animal model of human trisomy 21 (Down syndrome). However, the value of this model is limited to some extent because trisomy 16 mouse fetuses do not survive as live-born animals. Therefore, in an effort to produce viable mice with cells trisomic for chromosome 16, we have used an aggregation technique to generate trisomy 16 diploid (Ts 16 2n) chimeras. A total of 79 chimeric mice were produced, 11 of which were Ts 16 2n chimeras. Seven of these Ts 16 2n mice were analyzed as fetuses, just prior to birth, and 4 were analyzed as live-born animals. Unlike nonchimeric Ts 16 mouse fetuses which die shortly before birth with edema, congenital heart disease, and thymic and splenic hypoplasia, all but 1 of the Ts 16 2n animals were viable and phenotypically normal. The oldest of the live-born Ts 16 2n chimeras was 12 months old at the time of necropsy. Ts 16 cells, identified by coat color, enzyme marker, and/or karyotype analyses, comprised 50-60% of the brain, heart, lung, liver, and kidney in the 7 Ts 16 2n chimeric fetuses and 30-40% of these organs in the 4 live-born Ts 16 2n animals. Ts 16 cells comprised an average of 40% of the thymus and 80% of the spleen in the Ts 16 2n chimeras analyzed as fetuses, with no evidence of thymic or splenic hypoplasia. However, we observed a marked deficiency to Ts 16 cells in the blood, spleen, thymus, and bone marrow of live-born Ts 16 2n chimeras as compared to 2n 2n controls. These results demonstrate that although the Ts 16 2n chimeras were, with one exception, viable and phenotypically normal, each animal contained a significant proportion of trisomic cells in a variety of tissues, including the brain. Furthermore, our results suggest that although the abnormal development of Ts 16 thymus and spleen cells observed in Ts 16 fetuses is largely corrected in Ts 16 2n fetuses, Ts 16 erythroid and lymphoid cells have a severe proliferative disadvantage as compared to diploid cells in older live-born Ts 16 2n chimeras. Ts 16 2n chimeric mice will provide a valuable tool for studying the functional consequences of aneuploidy and may provide insight into the mechanisms by which trisomy 21 leads to developmental abnormalities in man.     

Chromosomal instability of mouse pluripotent cells cultured in vitro

The perspectives of using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs) in clinics makes the karyological analysis of these cells an important issue. In the present study, using methods of classical and molecular cytogenetics of chromosome, we carried out a karyological study of two mouse ES and two iPS cell lines derived de novo. We obsererved the X chromosome monosomy in all studied ES and iPS cell lines, which makes the modal number of chromosomes in these cell lines equal to 39. The chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of studied iPS. Our findings stress the importance of the careful cytogenetic evaluation of a pluripotent cell line, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.     

Double aneuploidy in three Egyptian patients: Down-Turner and Down-Klinefelter syndromes

The co-occurrence of two numerical chromosomal abnormalities in same individual (double aneuploidy) is relatively rare and its clinical presentations are variable depending on the predominating aneuploidy or a combination effect of both. Furthermore, double aneuploidy involving both autosomal and sex chromosomes is seldom described. In this study, we present three patients with double aneuploidy involving chromosome 21 and sex chromosomes. They all had the classical non disjunction trisomy 21; that was associated with monosomy X in two of them and double X in the other. Clinically, they had most of the phenotypic features of Down syndrome as well as variable features characteristic of Turner or Klinefelter syndrome. Cytogenetic studies and fluorescence in situ hybridization (FISH) analysis were carried out for all patients and their parents. The first patient was a male, mosaic with 2 cell lines (45,X/47,XY,+21) by regular banding techniques and had an affected sib with Down syndrome (47,XY,+21). The second was a female, mosaic (46,X,+21/47,XX,+21) where monosomy X was detected only by FISH in 15 percentages of cells, nevertheless, stigmata of Turner syndrome was more obvious in this patient. The third patient had non mosaic double trisomy; Down-Klinefelter (48,XXY,+21) presented with Down syndrome phenotype. Parental karyotypes and FISH studies for these patients were normal with no evidence of mosaicism. In this report, we review the variable clinical presentations among the few reported cases with the same aneuploidy in relation to ours. Also, the proposed mechanisms of double aneuploidy and the occurrence of non-disjunction in more than one family member are discussed. This study emphasizes the importance of molecular cytogenetics studies for more than one tissue in cases with atypical features of characteristic chromosomal aberration syndromes. To our knowledge, this is the first report of double aneuploidy, Down-Turner and Down-Klinefelter syndromes in Egyptian patients.     

Long-range chromosomal engineering is more efficient <Emphasis Type="Italic">in vitro</Emphasis> than <Emphasis Type="Italic">in vitro</Emphasis>

Cre/LoxP mediated chromosomal engineering in embryonic stem (ES) cells has a variety of applications, including the creation of model systems for studying aneuploidy. Targeted meiotic recombination (TAMERE) was proposed as a high efficiency in vivo alternative to effect Cre-mediated recombination, in which Cre recombinase under control of the Synaptonemal Complex 1 promoter is expressed during male meiosis in transgenic mice. TAMERE has been successfully used with LoxP sites up to 100 kb apart. We tested TAMERE for a chromosome engineering application in which LoxP sequences were integrated into sites 3.9 Mb apart on the same (cis) or opposite (trans) copies of mouse Chromosome 16 (MMU16). TAMERE was ineffective in generating either a deletion or a translocation in vivo. The TAMERE method may be of limited use for large genomic rearrangements. The desired translocation was achieved with an in vitro method that can be used in any ES cell line. Mice produced from the reciprocal duplication/deletion of MMU16 in a region homologous to human chromosome 21 provide models that are useful in studies of Down syndrome.     

Murine trisomy: developmental profiles of the embryo, and isolation of trisomic cellular systems

Many questions related to the development and the phenotypic expression of trisomy (Ts) are amenable to systematic investigation in a mouse model that allows the induction of Ts 1 to 19 by a breeding design of mice heterozygous for Robertsonian metacentric chromosomes. Some Ts do not survive the first critical phase of organogenesis on days 11 to 12 of fetal development; others as Ts 12, 14, 16, 18, and 19, have a life span until or beyond birth. Model type studies of the morphogenesis of developmental anomalies (e.g. craniocerebral, cardiovascular, or placental) are possible in Ts with a longer developmental span, and Ts 16 of the mouse is considered as a natural model of human trisomy 21. The eventual breakdown and death of the trisomic organism are inevitable. There is considerable interest to find ways for rescue and longer survival of Ts in competitive developmental systems, as e.g., in Ts in equilibrium with 2n blastocyst chimeras, or by isolation of trisomic cellular or tissue systems. Thus, the transfer of Ts hemopoietic stem cells of the fetal liver to irradiated adult recipients is a means of studying the functional capacities and maturation of trisomic hemopoiesis and lymphopoiesis. Both are almost completely restored by Ts 12, 14, 18, and 19 stem cell transplantation with survival periods of more than 6 months. But in other Ts, as of chromosomes 13 or 16, such capacity of reconstitution is impaired. The stepwise analysis of the effects of chromosome triplication on the cell level, in isolated functional systems and in the embryonic organism, is a promising way to understand the phenotypic expression of genome anomalies in complex developmental processes.     

BDNF and DYRK1A Are Variable and Inversely Correlated in Lymphoblastoid Cell Lines from Down Syndrome Patients

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.     

Genesis and systematization of cardiovascular anomalies and analysis of skeletal malformations in murine trisomy 16 and 19

Summary On account of genetic homologies, trisomy 16 in the mouse is generally regarded as a direct animal model of Down's syndrome. Mouse trisomy 19, on the other hand, can be seen as a general model of human trisomies. A detailed evaluation of the cardiovascular system and skeleton in 109 fetuses with trisomy 16 and 422 balanced siblings was carried out in order to systematize the cardiovascular anomalies and the pathogenetic mechanisms responsible for their formation according to (1) general retardation, (2) genetically determined impairment of neural-crest cell migration, and (3) direct gene action on organogenesis. Skeletal malformations in the form of a rib-vertebra syndrome encountered in Ts 16 are described here for the first time. In 108 fetuses and 219 neonates resulting from cross-breeding to induce trisomy 19, we found no significant increase in the frequency of the foregoing anomalies. These results are discussed with regard to a chromosome-specific genetic influence as opposed to a general effect of chromosome imbalance. The specificity of the Ts16 syndrome is compared with that of individual organ anomalies as can be induced by teratogenic agents. Our investigation shows that specific malformation patterns of a particular type can be produced by a variety of methods. However, the overall patterns of the two syndromes are highly chromosome-specific. On detailed examination, the malformation pattern of mouse trisomy 16 shows significant similarities with that of human trisomy 21.     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.     

Stem and progenitor cell dysfunction in human trisomies

Trisomy 21, the commonest constitutional aneuploidy in humans, causes profound perturbation of stem and progenitor cell growth, which is both cell context dependent and developmental stage specific and mediated by complex genetic mechanisms beyond increased Hsa21 gene dosage. While proliferation of fetal hematopoietic and testicular stem/progenitors is increased and may underlie increased susceptibility to childhood leukemia and testicular cancer, fetal stem/progenitor proliferation in other tissues is markedly impaired leading to the characteristic craniofacial, neurocognitive and cardiac features in individuals with Down syndrome. After birth, trisomy 21‐mediated premature aging of stem/progenitor cells may contribute to the progressive multi‐system deterioration, including development of Alzheimer's disease.