Unraveling the genetics of arsenic toxicity with cellular morphology QTL.

The health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Systemic, postsymptomatic antisense oligonucleotide rescues motor unit maturation delay in a new mouse model for type II/III spinal muscular atrophy.

Clinical presentation of spinal muscular atrophy (SMA) ranges from a neonatal-onset, very severe disease to an adult-onset, milder form. SMA is caused by the mutation of the Survival Motor Neuron 1 (SMN1) gene, and prognosis inversely correlates with the number of copies of the SMN2 gene, a human-specific homolog of SMN1. Despite progress in identifying potential therapies for the treatment of SMA, many questions remain including how late after onset treatments can still be effective and what the target tissues should be. These questions can be addressed in part with preclinical animal models; however, modeling the array of SMA severities in the mouse, which lacks SMN2, has proven challenging. We created a new mouse model for the intermediate forms of SMA presenting with a delay in neuromuscular junction maturation and a decrease in the number of functional motor units, all relevant to the clinical presentation of the disease. Using this new model, in combination with clinical electrophysiology methods, we found that administering systemically SMN-restoring antisense oligonucleotides (ASOs) at the age of onset can extend survival and rescue the neurological phenotypes. Furthermore, these effects were also achieved by administration of the ASOs late after onset, independent of the restoration of SMN in the spinal cord. Thus, by adding to the limited repertoire of existing mouse models for type II/III SMA, we demonstrate that ASO therapy can be effective even when administered after onset of the neurological symptoms, in young adult mice, and without being delivered into the central nervous system.

Cell morphology QTL reveal gene by environment interactions in a genetically diverse cell population.

Genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening as they offer power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia ­ structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis.

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states in vitro.

Mitotic chromosome alignment ensures mitotic fidelity by promoting interchromosomal compaction during anaphase.

Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell division; however, its importance to genomic stability and cellular fitness is not understood. Normal mammalian somatic cells lacking KIF18A function complete cell division without aligning chromosomes. These alignment-deficient cells display normal chromosome copy numbers in vitro and in vivo, suggesting that chromosome alignment is largely dispensable for maintenance of euploidy. However, we find that loss of chromosome alignment leads to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit, and the formation of micronuclei in vitro and in vivo. These defects slow cell proliferation and are associated with impaired postnatal growth and survival in mice. Our studies support a model in which the alignment of mitotic chromosomes promotes proper organization of chromosomes into a single nucleus and continued proliferation by ensuring that chromosomes segregate as a compact mass during anaphase.

Telenursing intervention increases psychiatric medication adherence in schizophrenia outpatients.

BACKGROUND: Promoting medication adherence is a critical issue in optimizing both physical and mental health in persons with schizophrenia. Average antipsychotic medication adherence is only 50%; few studies have examined nonpsychiatric medication adherence. Psychosocial interventions with components of problem solving and motivation have shown promise in improving adherence behaviors. OBJECTIVES: This study examines telephone intervention problem solving (TIPS) for outpatients with schizophrenia. TIPS is a weekly, provider-initiated, proactive telenursing intervention designed to help persons with schizophrenia respond to a variety of problems, including adherence problems. STUDY DESIGN: The authors completed objective measures of adherence to psychiatric and nonpsychiatric medications in 29 community-dwelling persons with schizophrenia, monthly for 3 months. STUDY RESULTS: Persons receiving TIPS had significantly higher objective adherence to psychiatric medications throughout the study period, F(1, 20) = 5.47, p = .0298. CONCLUSIONS: Clinicians should consider using TIPS as an adjunct to face-to-face appointments to support adherence in persons at risk. J Am Psychiatr Nurses Assoc, 2008; 14(3), 217-224. DOI: 10.1177/1078390308318750.