Dissection of a Down syndrome-associated trisomy to separate the gene dosage-dependent and -independent effects of an extra chromosome.

As an aneuploidy, trisomy is associated with mammalian embryonic and postnatal abnormalities. Understanding the underlying mechanisms involved in mutant phenotypes is broadly important and may lead to new strategies to treat clinical manifestations in individuals with trisomies, such as trisomy 21 [Down syndrome (DS)]. Although increased gene dosage effects because of a trisomy may account for the mutant phenotypes, there is also the possibility that phenotypic consequences of a trisomy can arise because of the presence of a freely segregating extra chromosome with its own centromere, i.e. a 'free trisomy' independent of gene dosage effects. Presently, there are no reports of attempts to functionally separate these two types of effects in mammals. To fill this gap, here we describe a strategy that employed two new mouse models of DS, Ts65Dn;Df(17)2Yey/+ and Dp(16)1Yey/Df(16)8Yey. Both models carry triplications of the same 103 human chromosome 21 gene orthologs; however, only Ts65Dn;Df(17)2Yey/+ mice carry a free trisomy. Comparison of these models revealed the gene dosage-independent impacts of an extra chromosome at the phenotypic and molecular levels for the first time. They are reflected by impairments of Ts65Dn;Df(17)2Yey/+ males in T-maze tests when compared with Dp(16)1Yey/Df(16)8Yey males. Results from the transcriptomic analysis suggest the extra chromosome plays a major role in trisomy-associated expression alterations of disomic genes beyond gene dosage effects. This model system can now be used to deepen our mechanistic understanding of this common human aneuploidy and obtain new insights into the effects of free trisomies in other human diseases such as cancers.

Triplications of human chromosome 21 orthologous regions in mice result in expansion of megakaryocyte-erythroid progenitors and reduction of granulocyte-macrophage progenitors.

Individuals with Down syndrome (DS) frequently have hematopoietic abnormalities, including transient myeloproliferative disorder and acute megakaryoblastic leukemia which are often accompanied by acquired GATA1 mutations that produce a truncated protein, GATA1s. The mouse has been used for modeling DS based on the syntenic conservation between human chromosome 21 (Hsa21) and three regions in the mouse genome located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. To assess the impact of the dosage increase of Hsa21 gene orthologs on the hematopoietic system, we characterized the related phenotype in the Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+ model which carries duplications spanning the entire Hsa21 orthologous regions on Mmu10, Mmu16 and Mmu17, and the Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+;Gata1Yeym2 model which carries a Gata1s mutation we engineered. Both models exhibited anemia, macrocytosis, and myeloproliferative disorder. Similar to human DS, the megakaryocyte-erythrocyte progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs) were significantly increased and reduced, respectively, in both models. The subsequent identification of all the aforementioned phenotypes in the Dp(16)1Yey/+ model suggests that the causative dosage sensitive gene(s) are in the Hsa21 orthologous region on Mmu16. Therefore, we reveal here for the first time that the human trisomy 21-associated major segmental chromosomal alterations in mice can lead to expanded MEP and reduced GMP populations, mimicking the dynamics of these myeloid progenitors in DS. These models will provide the critical systems for unraveling the molecular and cellular mechanism of DS-associated myeloproliferative disorder, and particularly for determining how human trisomy 21 leads to expansion of MEPs as well as how such an alteration leads to myeloproliferative disorder.

Analysis of mutations in primary and metastatic synovial sarcoma.

Synovial sarcoma is the most common pediatric non-rhabdomyosarcoma soft tissue sarcoma and accounts for about 8-10% of all soft tissue sarcoma in childhood and adolescence. The presence of a chromosomal translocation-associated SS18-SSX-fusion gene is causally linked to development of primary synovial sarcoma. Metastases occur in approximately 50-70% of synovial sarcoma cases with yet unknown mechanisms, which led to about 70-80% mortality rate in five years. To explore the possibilities to investigate metastatic mechanisms of synovial sarcoma, we carried out the first genome-wide search for potential genetic biomarkers and drivers associated with metastasis by comparative mutational profiling of 18 synovial sarcoma samples isolated from four patients carrying the primary tumors and another four patients carrying the metastatic tumors through whole exome sequencing. Selected from the candidates yielded from this effort, we examined the effect of the multiple missense mutations of ADAM17, which were identified solely in metastatic synovial sarcoma. The mutant alleles as well as the wild-type control were expressed in the mammalian cells harboring the SS18-SSX1 fusion gene. The ADAM17-P729H mutation was shown to enhance cell migration, a phenotype associated with metastasis. Therefore, like ADAM17-P729H, other mutations we identified solely in metastatic synovial sarcoma may also have the potential to serve as an entry point for unraveling the metastatic mechanisms of synovial sarcoma.

Trans-acting epigenetic effects of chromosomal aneuploidies: lessons from Down syndrome and mouse models.

An important line of postgenomic research seeks to understand how genetic factors can influence epigenetic patterning. Here we review epigenetic effects of chromosomal aneuploidies, focusing on findings in Down syndrome (DS, trisomy 21). Recent work in human DS and mouse models has shown that the extra chromosome 21 acts in trans to produce epigenetic changes, including differential CpG methylation (DS-DM), in specific sets of downstream target genes, mostly on other chromosomes. Mechanistic hypotheses emerging from these data include roles of chromosome 21-linked methylation pathway genes (DNMT3L and others) and transcription factor genes (RUNX1, OLIG2, GABPA, ERG and ETS2) in shaping the patterns of DS-DM. The findings may have broader implications for trans-acting epigenetic effects of chromosomal and subchromosomal aneuploidies in other human developmental and neuropsychiatric disorders, and in cancers.

Mouse-based genetic modeling and analysis of Down syndrome.

INTRODUCTION: Down syndrome (DS), caused by human trisomy 21 (Ts21), can be considered as a prototypical model for understanding the effects of chromosomal aneuploidies in other diseases. Human chromosome 21 (Hsa21) is syntenically conserved with three regions in the mouse genome. SOURCES OF DATA: A review of recent advances in genetic modeling and analysis of DS. Using Cre/loxP-mediated chromosome engineering, a substantial number of new mouse models of DS have recently been generated, which facilitates better understanding of disease mechanisms in DS. AREAS OF AGREEMENT: Based on evolutionary conservation, Ts21 can be modeled by engineered triplication of Hsa21 syntenic regions in mice. The validity of the models is supported by the exhibition of DS-related phenotypes. AREAS OF CONTROVERSY: Although substantial progress has been made, it remains a challenge to unravel the relative importance of specific candidate genes and molecular mechanisms underlying the various clinical phenotypes. GROWING POINTS: Further understanding of mechanisms based on data from mouse models, in parallel with human studies, may lead to novel therapies for clinical manifestations of Ts21 and insights to the roles of aneuploidies in other developmental disorders and cancers.

Human chromosome 21 orthologous region on mouse chromosome 17 is a major determinant of Down syndrome-related developmental cognitive deficits.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of developmental cognitive deficits, and the so-called Down syndrome critical region (DSCR) has been proposed as a major determinant of this phenotype. The regions on human chromosome 21 (Hsa21) are syntenically conserved on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. DSCR is conserved between the Cbr1 and Fam3b genes on Mmu16. Ts65Dn mice carry three copies of ∼100 Hsa21 gene orthologs on Mmu16 and exhibited impairments in the Morris water maze and hippocampal long-term potentiation (LTP). Converting the Cbr1-Fam3b region back to two copies in Ts65Dn mice rescued these phenotypes. In this study, we performed similar conversion of the Cbr1-Fam3b region in Dp(16)1Yey/+ mice that is triplicated for all ∼115 Hsa21 gene orthologs on Mmu16, which also resulted in the restoration of the wild-type phenotypes in the Morris water maze and hippocampal LTP. However, converting the Cbr1-Fam3b region back to two copies in a complete model, Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+, failed to yield the similar phenotypic restorations. But, surprisingly, converting both the Cbr1-Fam3b region and the Hsa21 orthologous region on Mmu17 back to two copies in the complete model did completely restore these phenotypes to the wild-type levels. Our results demonstrated that the Hsa21 orthologous region on Mmu17 is a major determinant of DS-related developmental cognitive deficits. Therefore, the inclusion of the three copies of this Hsa21 orthologous region in mouse models is necessary for unraveling the mechanism underlying DS-associated developmental cognitive deficits and for developing effective interventions for this clinical manifestation.

Engineered chromosome-based genetic mapping establishes a 3.7 Mb critical genomic region for Down syndrome-associated heart defects in mice.

Trisomy 21 (Down syndrome, DS) is the most common human genetic anomaly associated with heart defects. Based on evolutionary conservation, DS-associated heart defects have been modeled in mice. By generating and analyzing mouse mutants carrying different genomic rearrangements in human chromosome 21 (Hsa21) syntenic regions, we found the triplication of the Tiam1-Kcnj6 region on mouse chromosome 16 (Mmu16) resulted in DS-related cardiovascular abnormalities. In this study, we developed two tandem duplications spanning the Tiam1-Kcnj6 genomic region on Mmu16 using recombinase-mediated genome engineering, Dp(16)3Yey and Dp(16)4Yey, spanning the 2.1 Mb Tiam1-Il10rb and 3.7 Mb Ifnar1-Kcnj6 regions, respectively. We found that Dp(16)4Yey/+, but not Dp(16)3Yey/+, led to heart defects, suggesting the triplication of the Ifnar1-Kcnj6 region is sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16)4Yey/+ embryos showed that the Hsa21 gene orthologs located within the duplicated interval were expressed at the elevated levels, reflecting the consequences of the gene dosage alterations. Therefore, we have identified a 3.7 Mb genomic region, the smallest critical genomic region, for DS-associated heart defects, and our results should set the stage for the final step to establish the identities of the causal gene(s), whose elevated expression(s) directly underlie this major DS phenotype.

Ugt1a is required for the protective effect of selenium against irinotecan-induced toxicity.

PURPOSE: Irinotecan (CPT-11) is widely used for the treatment of patients with colorectal cancer. However, the adverse effects associated with the treatment have hindered the efficacies of irinotecan. We have shown that organic selenium compounds could significantly attenuate irinotecan-associated toxicity and enhance antitumor activity in xenograft tumor models. The objective of this study is to determine the role of a specific group of uridine diphosphate glucuronosyltransferases, which is coded by UGT1A, in detoxification process of irinotecan as well as selenium-associated protective effect against irinotecan-induced toxicity. METHODS: In this study, the toxicities of irinotecan, docetaxel and cisplatin in the Ugta1 mutant rats and their wild-type controls were compared. The plasma concentrations of irinotecan and SN-38 were measured. The modulatory effect of a selenium compound on irinotecan-induced toxicity was analyzed in these rats. RESULTS: We demonstrated that the maximum tolerated doses of irinotecan in the homozygous mutant rats were significantly lower than those in wild-type rats, 25 mg/kg × 1 versus 200 mg/kg × 1 and 3 mg/kg/day × 3 versus 100 mg/kg/day × 3, respectively. The enhanced sensitivity was specific to irinotecan and was not observed with other chemotherapeutic agents, such as docetaxel and cisplatin, where Ugt1a is not required for their metabolism. Our results also showed that selective protection against irinotecan-induced toxicity by 5-methylselenocysteine was achieved in the wild-type rats but not in the Ugt1a null rats. CONCLUSION: These data support the hypothesis that expression of UGT1A is critical for 5-methylselenocysteine to exert its protective effect against irinotecan-induced toxicity.

Genetic analysis of Down syndrome facilitated by mouse chromosome engineering.

Human trisomy 21 is the most frequent live-born human aneuploidy and causes a constellation of disease phenotypes classified as Down syndrome, which include heart defects, myeloproliferative disorder, cognitive disabilities and Alzheimer-type neurodegeneration. Because these phenotypes are associated with an extra copy of a human chromosome, the genetic analysis of Down syndrome has been a major challenge. To complement human genetic approaches, mouse models have been generated and analyzed based on evolutionary conservation between the human and mouse genomes. These efforts have been greatly facilitated by Cre/loxP-mediated mouse chromosome engineering, which may result in the establishment of minimal critical genomic regions and eventually new dosage-sensitive genes associated with Down syndrome phenotypes. The success in genetic analysis of Down syndrome will further enhance our understanding of this disorder and lead to better strategies in developing effective therapeutic interventions.

Genetic analysis of Down syndrome-associated heart defects in mice.

Human trisomy 21, the chromosomal basis of Down syndrome (DS), is the most common genetic cause of heart defects. Regions on human chromosome 21 (Hsa21) are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this study, we have analyzed the impact of duplications of each syntenic region on cardiovascular development in mice and have found that only the duplication on Mmu16, i.e., Dp(16)1Yey, is associated with heart defects. Furthermore, we generated two novel mouse models carrying a 5.43-Mb duplication and a reciprocal deletion between Tiam1 and Kcnj6 using chromosome engineering, Dp(16Tiam1-Kcnj6)Yey/+ and Df(16Tiam1-Kcnj6)Yey/+, respectively, within the 22.9-Mb syntenic region on Mmu16. We found that Dp(16Tiam1-Kcnj6)Yey/+, but not Dp(16)1Yey/Df(16Tiam1-Kcnj6)Yey, resulted in heart defects, indicating that triplication of the Tiam1-Knj6 region is necessary and sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16Tiam1-Kcnj6)Yey/+ embryos confirmed elevated expression levels for the genes located in the Tiam-Kcnj6 region. Therefore, we established the smallest critical genomic region for DS-associated heart defects to lay the foundation for identifying the causative gene(s) for this phenotype.

Increased insulin action in SKIP heterozygous knockout mice.

Insulin controls glucose homeostasis and lipid metabolism, and insulin impairment plays a critical role in the pathogenesis of diabetes mellitus. Human skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) is a member of the phosphatidylinositol 3,4,5-trisphosphate phosphatase family (T. Ijuin et al. J. Biol. Chem. 275:10870-10875, 2000; T. Ijuin and T. Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). Previous studies showed that SKIP negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling (Ijuin and Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). We now have generated mice with a targeted mutation of the mouse ortholog of the human SKIP gene, Pps. Adult heterozygous Pps mutant mice show increased insulin sensitivity and reduced diet-induced obesity with increased Akt/protein kinase B (PKB) phosphorylation in skeletal muscle but not in adipose tissue. The insulin-induced uptake of 2-deoxyglucose into the isolated soleus muscle was significantly enhanced in Pps mutant mice. A hyperinsulinemic-euglycemic clamp study also revealed a significant increase in the rate of systemic glucose disposal in Pps mutant mice without any abnormalities in hepatic glucose production. Furthermore, in vitro knockdown studies in L6 myoblast cells revealed that reduction of SKIP expression level increased insulin-stimulated Akt/PKB phosphorylation and 2-deoxyglucose uptake. These results imply that SKIP regulates insulin signaling in skeletal muscle. Thus, SKIP may be a promising pharmacologic target for the treatment of insulin resistance and diabetes.