Human Aneuploidy Research Articles

Computational investigation of pathogenic nsSNPs in CEP63 protein

Centrosomes are central regulators of mitosis that are often amplified in cancer cells. Centrosomal protein of 63kDa (CEP63) is a centrosomal protein that has an effective role in mitotic spindle assembly and cell cycle regulation. Genetic alterations in CEP63 coding gene has been widely studied for inducing aneuploidy and solid tumors in humans. The nonsynonymous single nucleotide polymorphisms (nsSNPs) are a genetic variant resulting in amino acid substitution and are reported in a wide range of human diseases. Here we report one new SNP (rs112926188) in a CEP63 coding region that can potentially disrupt the structure and basic functionality of a CEP63 protein. We used extensive functional and structural level analyses of an available SNP in a CEP63 coding gene. Furthermore the disease-association analysis was carried out to examine the possible pathogenic variant among the available dataset. To understand atomic arrangement in 3D space, native and pathogenic mutant structures were modeled. Molecular dynamics simulations were performed to understand structural consequences of prioritized deleterious mutation. Our analysis showed that rs112926188 allele substituting proline at the 61st residue position (L61P) produced more flexibility in 3D space. Moreover the flexible nature of mutant L61P was validated by a hydrogen bond network. This nature of mutant L61P CEP63 may restrict the recruitment of essential centrosomal proteins to their respective location and may play an active role in inducing aneuploidy.

Abstract 901: Mitotic kinase inhibitors synergize with PI3K and Akt inhibitors in a novel model of anaplastic thyroid carcinomas

Abstract Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer, and often derives from pre-existing well-differentiated tumors. Despite a relatively low prevalence, it accounts for a disproportionate number of thyroid cancer-related deaths, due to its resistance to any therapeutic approach. We have generated a mouse model in which the simultaneous selective activation of the PI3K pathway and inactivation of p53 in the thyroid epithelial cells leads to the development of ATCs. These tumors display all the features of their human counterpart, including pleomorphism, epithelial-mesenchymal transition, aneuploidy, local invasion and distant metastases. Expression profiling of the murine ATCs reveals a significant overlap with genes found deregulated in human ATC, including genes involved in mitosis control, such as Aurora kinase A (AURKA), its co-activator TPX2, and Polo like kinase-1 (PLK1). These proteins are crucial driving forces for normal mitotic spindle formation, centrosome maturation and separation, and their overexpression has been demonstrated in a wide range of tumors. MLN8237 (AURKA inhibitor), TAK960 and GSK461364 (PLK1 inhibitors) are a promising new generation of anti-mitotic agents. We show that these molecules inhibit cell proliferation and induce cell death in mouse ATC-derived cell lines and in the human ATC cell lines CAL62 and C643. A dose-dependent decrease in the degree of chromosome alignment and spindle bipolarity during mitosis is observed after the treatments, together with a change in mitotic index indicated by the decrease of the mitotic marker pHisH3. FACS analysis reveals a G2/M phase arrest followed by apoptosis. Furthermore, small molecule inhibitors of PI3K or AKT potently synergize with these anti-mitotic compounds. Taken together these data suggest that AURKA and PLK1 inhibitors are promising therapeutic agents against undifferentiated thyroid carcinoma cells, and that concurrent inhibition of key driver pathways is a viable option to increase efficacy in this otherwise invariably lethal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 901. doi:1538-7445.AM2012-901

Mammalian X chromosome inactivation evolved as a dosage-compensation mechanism for dosage-sensitive genes on the X chromosome

How and why female somatic X-chromosome inactivation (XCI) evolved in mammals remains poorly understood. It has been proposed that XCI is a dosage-compensation mechanism that evolved to equalize expression levels of X-linked genes in females (2X) and males (1X), with a prior twofold increase in expression of X-linked genes in both sexes ("Ohno's hypothesis"). Whereas the parity of X chromosome expression between the sexes has been clearly demonstrated, tests for the doubling of expression levels globally along the X chromosome have returned contradictory results. However, changes in gene dosage during sex-chromosome evolution are not expected to impact on all genes equally, and should have greater consequences for dosage-sensitive genes. We show that, for genes encoding components of large protein complexes (≥ 7 members)--a class of genes that is expected to be dosage-sensitive--expression of X-linked genes is similar to that of autosomal genes within the complex. These data support Ohno's hypothesis that XCI acts as a dosage-compensation mechanism, and allow us to refine Ohno's model of XCI evolution. We also explore the contribution of dosage-sensitive genes to X aneuploidy phenotypes in humans, such as Turner (X0) and Klinefelter (XXY) syndromes. X aneuploidy in humans is common and is known to have mild effects because most of the supernumerary X genes are inactivated and not affected by aneuploidy. Only genes escaping XCI experience dosage changes in X-aneuploidy patients. We combined data on dosage sensitivity and XCI to compute a list of candidate genes for X-aneuploidy syndromes.

DNA and ability to reproduce: the ‘Secret’ of evolution

DNA and ability to reproduce: the ‘Secret’ of evolution

Morphological integration of soft-tissue facial morphology in down syndrome and siblings

Down syndrome (DS), resulting from trisomy of chromosome 21, is the most common live-born human aneuploidy. The phenotypic expression of trisomy 21 produces variable, though characteristic, facial morphology. Although certain facial features have been documented quantitatively and qualitatively as characteristic of DS (e.g., epicanthic folds, macroglossia, and hypertelorism), all of these traits occur in other craniofacial conditions with an underlying genetic cause. We hypothesize that the typical DS face is integrated differently than the face of non-DS siblings, and that the pattern of morphological integration unique to individuals with DS will yield information about underlying developmental associations between facial regions. We statistically compared morphological integration patterns of immature DS faces (N = 53) with those of non-DS siblings (N = 54), aged 6-12 years using 31 distances estimated from 3D coordinate data representing 17 anthropometric landmarks recorded on 3D digital photographic images. Facial features are affected differentially in DS, as evidenced by statistically significant differences in integration both within and between facial regions. Our results suggest a differential affect of trisomy on facial prominences during craniofacial development.

The Origin of Aneuploidy in Humans: Does Prophase Set the Stage?

The Origin of Aneuploidy in Humans: Does Prophase Set the Stage?

Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans

IntroductionAneuploidy (the presence of extra or missing chromosomes) arises primarily through chromosome segregation errors in the oocyte at meiosis I but the details of mechanism by which such errors occur...

Deficit of complex I activity in human skin fibroblasts with chromosome 21 trisomy and overproduction of reactive oxygen species by mitochondria: involvement of the cAMP/PKA signalling pathway

DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.

MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13

Many human aneuploidy syndromes have unique phenotypic consequences, but in most instances it is unclear whether these phenotypes are attributable to alterations in the dosage of specific genes. In human trisomy 13, there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. Using partial trisomy cases, we mapped this trait to chromosomal band 13q14; by examining the genes in this region, two microRNAs, miR-15a and -16-1, appear as top candidates for the elevated HbF levels. Indeed, increased expression of these microRNAs in primary human erythroid progenitor cells results in elevated fetal and embryonic hemoglobin gene expression. Moreover, we show that a direct target of these microRNAs, MYB, plays an important role in silencing the fetal and embryonic hemoglobin genes. Thus we demonstrate how the developmental regulation of a clinically important human trait can be better understood through the genetic and functional study of aneuploidy syndromes and suggest that miR-15a, -16-1, and MYB may be important therapeutic targets to increase HbF levels in patients with sickle cell disease and β-thalassemia.

Morphometry of the Ear in North Sudanese Subjects With Down Syndrome

Down syndrome (DS) is the most frequent live-born autosomal aneuploidy in humans. Scanty data on the craniofacial phenotype of African subjects with DS have been published so far. We wanted to detail the morphologic characteristics of the ears in north Sudanese subjects with DS. The three-dimensional coordinates of 13 soft-tissue landmarks on the ears were obtained using a laser scanner in 64 north Sudanese subjects with DS aged 4 to 34 years and in 682 sex- and age-matched control subjects. From the landmarks, left and right linear distances (ear width and length), ratios (ear width-to-ear length), areas (ear area), angles (angle of the auricle vs the facial midplane), and the three-dimensional symmetry index were calculated. Distances, angles, areas, and ratios were computed. Subject and reference data were compared by computing z scores and calculating Student t tests. Ear width, length, and area were significantly (Student t test, P < 0.001) smaller in the subjects with DS than in the reference subjects. On the right side of the face, the subjects with DS had larger ear width-to-ear length ratios and larger angles of the auricle versus the facial midplane than the reference subjects. The three-dimensional symmetry index was significantly larger in the reference subjects. In conclusion, ear dimensions, position, and shape significantly differed in subjects with DS when compared with sex-, age-, and ethnic group-matched control subjects.

Transcriptomic analysis of cell-free fetal RNA suggests a specific molecular phenotype in trisomy 18.

Trisomy 18 is a common human aneuploidy that is associated with significant perinatal mortality. Unlike the well-characterized "critical region" in trisomy 21 (21q22), there is no corresponding region on chromosome 18 associated with its pathogenesis. The high morbidity and mortality of affected individuals has limited extensive investigations. In order to better understand the molecular mechanisms underlying the congenital anomalies observed in this condition, we investigated the in utero gene expression profile of second trimester fetuses affected with trisomy 18. Total RNA was extracted from cell-free amniotic fluid supernatant from aneuploid fetuses and euploid controls matched for gestational age and hybridized to Affymetrix U133 Plus 2.0 arrays. Individual differentially expressed transcripts were obtained by two-tailed t tests. Over-represented functional pathways among these genes were identified with DAVID and Ingenuity(®) Pathways Analysis. Results show that three hundred and fifty-two probe sets representing 251 annotated genes were statistically significantly differentially expressed between trisomy 18 and controls. Only 7 genes (2.8% of the annotated total) were located on chromosome 18, including ROCK1, an up-regulated gene involved in valvuloseptal and endocardial cushion formation. Pathway analysis indicated disrupted function in ion transport, MHCII/T cell mediated immunity, DNA repair, G-protein mediated signaling, kinases, and glycosylation. Significant down-regulation of genes involved in adrenal development was identified, which may explain both the abnormal maternal serum estriols and the pre and postnatal growth restriction in trisomy 18. Comparison of this gene set to one previously generated for trisomy 21 fetuses revealed only six overlapping differentially regulated genes. This study contributes novel information regarding functional developmental gene expression differences in fetuses with trisomy 18.

Phenotypic Consequences of Aneuploidy inArabidopsis thaliana

Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.

Advances in understanding the genetic causes and mechanisms of female germ cell aneuploidy

Aneuploidy, the gain or loss of one or more chromosomes, is the main ascertained cause of embryonic loss and the most common genetic defect in newborns. Human germ cell aneuploidy is characterized by its more frequent occurrence during oogenesis and its correlation with maternal age. Although we still lack a unique precept explaining the etiology of aneuploidy, progress has been made in elucidating the mechanisms that control chromosome segregation. The use of experimental models with genetic mutations in specific genes has helped to identify mechanisms that predispose female germ cells to aneuploidy, including disturbances in meiotic recombination, spindle attachment and cell cycle checkpoint. This article presents the latest advances in identifying genetic factors involved in the genesis of germ cell aneuploidy.

Aneuploid Human Oocytes and Embryos Possess Significantly Reduced Quantities of Telomere DNA.

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading cause of miscarriage and developmental delay in newborns. Although maternal age is a well known risk factor, the underlying molecular etiology of human aneuploidy is unknown. Since telomere DNA content is associated with genomic instability and is naturally reset during mammalian preimplantation embryonic development, telomere DNA quantity may play a role in the development of aneuploidy commonly found in human embryos. To begin to characterize the potential association of telomere biology with development of human aneuploidy, we have characterized relative telomere DNA quantity at various stages of human preimplantation embryogenesis. First, we developed a method capable of accurate quantitation of telomere DNA from single cells. Whole genome amplification (WGA) of picogram amounts of DNA from cells with previously established telomere DNA quantities provided results equivalent to those obtained using large quantities of purified total DNA (Pearson correlation coefficient=0.97). This technology was then applied to human oocytes and embryos. Excess WGA DNA was available for telomere DNA analysis of oocyte and embryo biopsies as a result of previously performing SNP microarray based 24 chromosome aneuploidy screening on human in vitro fertilization derived samples. Paired analysis of aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. Embryo fragmentation rates, which were previously shown to correlate with telomere DNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Results demonstrated, for the first time, that aneuploid human polar bodies possess significantly less telomere DNA than euploid polar bodies (-3.07 fold, P=0.016). Since the first and second polar bodies are extruded upon completion of meiosis I and II, respectively, and reflect the chromosomal complement of the remaining oocyte, abnormalities observed in polar bodies are indicative of development of aneuploidy during meiosis of the oocyte. Therefore, the present results indicate that oocytes with reduced telomere DNA quantity are prone to development of aneuploidy during meiosis. We also show that aneuploid embryonic cells possess significantly less telomere DNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blastocyst stage (-1.18 fold, P=0.340). These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation between the cleavage and blastocyst stages of development. This further suggests that aneuploidy is not associated with failed resetting of telomere DNA length but is instead associated with failed telomere maintenance in the maternal genome during oogenesis and early embryo development. (platform)

Noninvasive prenatal chromosomal aneuploidy detection using plasma cell-free nucleic acid

It has been shown that by the end of the first trimester of pregnancy, a small but significant portion of DNA extracted from maternal plasma is of fetal origin. This knowledge has been utilized for the development of clinical diagnostic assays that are widely used outside the USA for the minimally invasive prenatal determination of, for example, fetal Rhesus antigen status. Such assays that enable the detection of paternally inherited fetal alleles are relatively straightforward, because the fetal sequence of interest is unique to the father and is therefore not masked by any identical maternal alleles. By contrast, maternally inherited traits, including the majority of common human aneuploidies such as trisomy 21, are much harder to detect in plasma because of the overwhelming background of endogenous maternal sequences that are identical to the fetal target alleles. One way to ‘see’ fetal DNA past the veil of maternal genomic DNA is to utilize methods that can selectively amplify or enrich fetal alleles...

Chromosome 21 non-disjunction and Down syndrome birth in an Indian cohort: analysis of incidence and aetiology from family linkage data

We analysed the family linkage data obtained from short tandem repeat (STR) genotyping of 212 unrelated Indian families having a single Down syndrome (DS) baby each, in order to explore the incidence and aetiology of this human aneuploidy in our cohort. The estimated values of maternal meiotic I and meiotic II non-disjunction (NDJ) errors of chromosome 21 (Ch 21) were approximately 78 and approximately 22%, respectively. Within the paternal outcome group, about 47 and 53% were accounted for NDJ at meiosis I and meiosis II, respectively. We estimated only approximately 2% post-zygotic mitotic errors. The comparison of average age of conception between controls and DS-bearing mothers revealed a significant difference (P<0.001) with DS-bearing women were on an average older than controls and meiotic II non-disjoined mothers were oldest among meiotic outcome groups. Our linkage analysis suggested an overall reduction in recombination by more than 50% on meiotic I non-disjoined maternal Ch 21 with error prone to susceptible chiasma formation within the approximately 5.1 kbp segment near the telomeric end. We stratified meiotic I non-disjoined women in three age groups, viz. young (<or=28 years), middle (29-34 years) and old (>or=35 years) and found linear decrease in the frequency of achiasmate meiosis from the young to the old group. In contrary, a linear increase in the multiple chiasma frequency from the young to the old group was observed. Considering these results together, we propose that the risk factors for Ch 21 NDJ are of two types, one being 'maternal age-independent' and the other being 'maternal age-dependent'. Moreover, a comparison of our present Indian dataset with that of other published data of ethnically different populations suggested that the genetics that underlies the NDJ of Ch 21 is probably universal irrespective of racial difference across human populations. The present study is the first population-based report on any DS cohort from the Indian subcontinent and our work will help future workers in understanding better the aetiology of this birth defect.

C29 The origin of aneuploidy in humans: where we've been, where we're going

C29 The origin of aneuploidy in humans: where we've been, where we're going

Abstract 4391: 4-Hydroxy-2-nonenal causes tetraploidy through a macrophage-induced bystander effect triggered by a commensal bacterium

Abstract Tetraploidy can be an intermediate condition for cells undergoing tranformation from diploidy to aneuploidy. We previously showed that Enterococcus faecalis, a human intestinal commensal, generated tetraploidy and aneuploidy in colonic epithelial cells via a macrophage-induced bystander effect. To explore potential mediators for this effect, supernatants from E. faecalis-infected macrophages were analyzed by HPLC coupled to a coulometric detection system. Significantly increased concentrations of 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids, were detected in macrophage supernatants. This system was used to purify macrophage-generated 4-HNE for further experiments. To investigate aneugenic effects of 4-HNE, a near-diploid human colon cancer cell line (HCT116) and non-transformed diploid mouse colonic epithelial cell line (YAMC) were exposed to 4-HNE and ploidy analyzed by FACS. For HCT116 cells the percent tetraploidy significantly increased following exposure to 4-HNE at 2.5 µM compared to untreated controls (9.90% ± 0.09 vs. 0.21% ± 0.08, P &amp;lt; 0.001). In comparison, the percent tetraploidy was increased to 0.68% ± 0.10 for YAMC cells exposed to 1 µM 4-HNE compared to 0.12% ± 0.06 for untreated controls (P &amp;lt; 0.001). This showed that 4-HNE generated tetraploidy in both transformed and non-transformed cells. To address potential mechanisms by which 4-HNE produced tetraploidy, HCT116 cells exposed to 4-HNE were stained with anti-α/β tubulin antibody and microtubules analyzed by confocal microscopy. We found cells exposed to 4-HNE had degraded microtubules and abnormal mitotic patterns, implicating the induction of tetraploidy through inhibition of microtubule assembly. Moreover, for HCT116 and YAMC cells, 4-HNE caused a G2/M cell cycle arrest with increased γH2AX foci suggesting DNA double-strand breaks. Next, we silenced mGSTα4-4, a key enzyme in the detoxification of 4-HNE, by constitutively expressing RNAi in cells. FACS analysis showed that γH2AX positivity remarkably increased in YAMC cells with silenced mGSTα4-4 that were exposed to E. faecalis-infected macrophages compared to non-infected macrophages. No changes were seen in wildtype YAMC cells. Similarly, γH2AX foci increased in mGSTα4-4-silenced YAMC cells exposed to 4-HNE compared to the wildtype control (41.8% ± 1.3 vs. 24.6% ± 6.5, P = 0.01). Finally, percent tetraploidy also significantly increased for mGSTα4-4-silenced YAMC cells exposed to 4-HNE, supporting the hypothesis that 4-HNE can specifically generate tetraploidy. In sum, E. faecalis-infected macrophages produce 4-HNE that, in turn, induces tetraploidy, DNA double-strand breaks and G2/M arrest in colonic epithelial cells. Inactivation of mGSTα4-4, a detoxifying enzyme for 4-HNE, exacerbated these effects. We propose 4-HNE as a mediator for the E. faecalis-infected macrophage bystander effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4391.

Letter to the Editor] PCR Prescreen for Genotyping the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (trisomy 21, or DS) is the most common live-born aneuploidy in humans, occurring in approximately 1 in 700 live births.

A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

BackgroundDosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21.ResultsTo elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis.ConclusionsWe determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders.