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Cell cycle dysregulation is a prerequisite for cancer formation. However, whether the type of cell cycle dysregulation event a cell incurs during transformation to malignancy influences the type of cancer that evolves or clinical outcome is unknown. In a comprehensive analysis of cell cycle dysregulation in breast cancer patient tumors, we associate mutations in each of four cell cycle checkpoint kinase genes, ATM, CHEK2, ATR and CHEK1, with known tumor characteristics and clinical outcome, and test these associations experimentally using transgenic mice, patient-derived xenografts and breast cancer cell line model systems. Results of this work demonstrate that dysregulation of specific cell cycle checkpoint kinases differently impacts the type of breast cancer that evolves in patients and in experimental model systems, and influences treatment responsiveness and disease progression. For instance, CHEK2 mutations associate preferentially with the incidence of metastatic, premenopausal estrogen receptor (ER)+/HER2- breast cancer in patient data (p=0.001) that is resistant to standard frontline therapy (HR=6.15, p=0.01). These associations appear causal when tested in an immune-competent genetically-engineered mouse model of Chk2 loss, in patient-derived xenograft, and in cell line experiments. On the other hand, ATR mutation by itself is not frequent in ER+/HER2- breast cancer, but co-incident mutation of ATR and TP53 is 2-fold enriched (p=0.002) and associates with metastatic progression (HR=2.01, p=0.007). Concordantly, ATR dysregulation induces metastatic phenotypes in ER+/HER- TP53 mutant, but not in TP53 wildtype, cell lines. Together, these results systematize the impact of individual cell cycle checkpoint kinases on the evolution of cancer subtypes, and on disease progression. Statement of Significance These findings reframe the paradigm of breast cancer classification through the lens of early cell cycle dysregulation events by demonstrating that cell cycle decisions during malignant transformation can direct the type of breast cancer that evolves, how it will respond to treatment, and whether it will metastasize. This work provides rationale for streamlined testing of checkpoint kinase dysregulation to improve precision diagnostics for cancer patients. Citation Format: Sinem Seker, Elena Oropeza, Sabrina Carrel, Aloran Mazumder, Nindo Punturi, Jonathan Lei, Meenakshi Anurag, Bora Lim, Matthew Bainbridge, Svasti Haricharan. Cell cycle dysregulation in breast cancer: why the details matter [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-10-02.

Newborn screening (NBS) dramatically improves outcomes in severe childhood disorders by treatment before symptom onset. In many genetic diseases, however, outcomes remain poor because NBS has lagged behind drug development. Rapid whole-genome sequencing (rWGS) is attractive for comprehensive NBS because it concomitantly examines almost all genetic diseases and is gaining acceptance for genetic disease diagnosis in ill newborns. We describe prototypic methods for scalable, parentally consented, feedback-informed NBS and diagnosis of genetic diseases by rWGS and virtual, acute management guidance (NBS-rWGS). Using established criteria and the Delphi method, we reviewed 457 genetic diseases for NBS-rWGS, retaining 388 (85%) with effective treatments. Simulated NBS-rWGS in 454,707UK Biobank subjects with 29,865 pathogenic or likely pathogenic variants associated with 388 disorders had a true negative rate (specificity) of 99.7% following root cause analysis. In 2,208 critically ill children with suspected genetic disorders and 2,168 of their parents, simulated NBS-rWGS for 388 disorders identified 104 (87%) of 119 diagnoses previously made by rWGS and 15 findings not previously reported (NBS-rWGS negative predictive value 99.6%, true positive rate [sensitivity] 88.8%). Retrospective NBS-rWGS diagnosed 15 children with disorders that had been undetected by conventional NBS. In 43 of the 104 children, had NBS-rWGS-based interventions been started on day of life 5, the Delphi consensus was that symptoms could have been avoided completely in seven critically ill children, mostly in 21, and partially in 13. We invite groups worldwide to refine these NBS-rWGS conditions and join us to prospectively examine clinical utility and cost effectiveness.

The blood–brain barrier ensures CNS homeostasis and protection from injury. Claudin-5 (CLDN5), an important component of tight junctions, is critical for the integrity of the blood–brain barrier. We have identified de novo heterozygous missense variants in CLDN5 in 15 unrelated patients who presented with a shared constellation of features including developmental delay, seizures (primarily infantile onset focal epilepsy), microcephaly and a recognizable pattern of pontine atrophy and brain calcifications. All variants clustered in one subregion/domain of the CLDN5 gene and the recurrent variants demonstrate genotype–phenotype correlations. We modelled both patient variants and loss of function alleles in the zebrafish to show that the variants analogous to those in patients probably result in a novel aberrant function in CLDN5. In total, human patient and zebrafish data provide parallel evidence that pathogenic sequence variants in CLDN5 cause a novel neurodevelopmental disorder involving disruption of the blood–brain barrier and impaired neuronal function.

Precision medicine strives for highly individualized treatments for disease under the notion that each individual's unique genetic makeup and environmental exposures imprints upon them not only a disposition to illness, but also an optimal therapeutic approach. In the realm of rare disorders, genetic predisposition is often the predominant mechanism driving disease presentation. For such, mostly, monogenic disorders, a causal gene to phenotype association is likely. As a result, it becomes important to query the patient's genome for the presence of pathogenic variations that are likely to cause the disease. Determining whether a variant is pathogenic or not is critical to these analyses and can be challenging, as many disease-causing variants are novel and, ergo, have no available functional data to help categorize them. This problem is exacerbated by the need for rapid evaluation of pathogenicity, since many genetic diseases present in young children who will experience increased morbidity and mortality without rapid diagnosis and therapeutics. Here, we discuss the utility of animal models, with a focus mainly on C. elegans, as a contrast to tissue culture and in silico approaches, with emphasis on how these systems are used in determining pathogenicity of variants with uncertain significance and then used to screen for novel therapeutics.

ImportanceUnderstanding the causes of infant mortality shapes public health, surveillance, and research investments. However, the association of single-locus (mendelian) genetic diseases with infant mortality is poorly understood.ObjectiveTo determine the association of genetic diseases with infant mortality.Design, Setting, and ParticipantsThis cohort study was conducted at a large pediatric hospital system in San Diego County (California) and included 546 infants (112 infant deaths [20.5%] and 434 infants [79.5%] with acute illness who survived; age, 0 to 1 year) who underwent diagnostic whole-genome sequencing (WGS) between January 2015 and December 2020. Data analysis was conducted between 2015 and 2022.ExposureInfants underwent WGS either premortem or postmortem with semiautomated phenotyping and diagnostic interpretation.Main Outcomes and MeasuresProportion of infant deaths associated with single-locus genetic diseases.ResultsAmong 112 infant deaths (54 girls [48.2%]; 8 [7.1%] African American or Black, 1 [0.9%] American Indian or Alaska Native, 8 [7.1%] Asian, 48 [42.9%] Hispanic, 1 [0.9%] Native Hawaiian or Pacific Islander, and 34 [30.4%] White infants) in San Diego County between 2015 and 2020, single-locus genetic diseases were the most common identifiable cause of infant mortality, with 47 genetic diseases identified in 46 infants (41%). Thirty-nine (83%) of these diseases had been previously reported to be associated with childhood mortality. Twenty-eight death certificates (62%) for 45 of the 46 infants did not mention a genetic etiology. Treatments that can improve outcomes were available for 14 (30%) of the genetic diseases. In 5 of 7 infants in whom genetic diseases were identified postmortem, death might have been avoided had rapid, diagnostic WGS been performed at time of symptom onset or regional intensive care unit admission.Conclusions and RelevanceIn this cohort study of 112 infant deaths, the association of genetic diseases with infant mortality was higher than previously recognized. Strategies to increase neonatal diagnosis of genetic diseases and immediately implement treatment may decrease infant mortality. Additional study is required to explore the generalizability of these findings and measure reduction in infant mortality.

White dwarfs are useful objects with which to study the local interstellar medium (ISM). High ionisation state absorption features that cannot be attributed to the photosphere or the ISM have been observed along the line-of-sight to a number of white dwarf stars. Suggested origins of these lines include ionisation from past supernovae, stellar winds, circumstellar disks, photoionisation from nearby hot stars or also from the white dwarf itself. In this study we consider the origin of these non-photospheric highly ionised lines in two stars towards a rarefied region of the galaxy known as the extended β CMa Tunnel. We present preliminary results from our analysis of the first of these two stars.

Observations of the redshift z=7.085 quasar J1120+0641 have been used to search for variations of the fine structure constant, alpha, over the redshift range 5.5 to 7.1. Observations at z=7.1 probe the physics of the universe when it was only 0.8 billion years old. These are the most distant direct measurements of alpha to date and the first measurements made with a near-IR spectrograph. A new AI analysis method has been employed. Four measurements from the X-SHOOTER spectrograph on the European Southern Observatory's Very Large Telescope (VLT) directly constrain any changes in alpha relative to the value measured on Earth (alpha_0). The weighted mean strength of the electromagnetic force over this redshift range in this location in the universe is da/a = (alpha_z - alpha_0)/alpha_0 = (-2.18 +/- 7.27) X 10^{-5}, i.e. we find no evidence for a temporal change from the 4 new very high redshift measurements. When the 4 new measurements are combined with a large existing sample of lower redshift measurements, a new limit on possible spatial variation of da/a is marginally preferred over a no-variation model at the 3.7 sigma level., Comment: Accepted for publication in Science Advances

Using newly obtained high-resolution data ($R\sim{1\times{10}^{5}}$) from the \textit{Hubble Space Telescope}, and archival UV data from the \textit{Far Ultraviolet Spectroscopic Explorer} we have conducted a detailed UV survey of the three hot, metal-polluted white dwarfs WD0455-282, WD0621-376, and WD2211-495. Using bespoke model atmospheres we measured $T_{\mathrm{eff}}$, log $g$, and photospheric abundances for these stars. In conjunction with data from Gaia we measured masses, radii, and gravitational redshift velocities for our sample of objects. We compared the measured photospheric abundances with those predicted by radiative levitation theory, and found that the observed Si abundances in all three white dwarfs, and the observed Fe abundances in WD0621-376 and WD2211-495, were larger than those predicted by an order of magnitude. These findings imply not only an external origin for the metals, but also ongoing accretion, as the metals not supported by radiative levitation would sink on extremely short timescales. We measured the radial velocities of several absorption features along the line of sight to the three objects in our sample, allowing us to determine the velocities of the photospheric and interstellar components along the line of sight for each star. Interestingly, we made detections of circumstellar absorption along the line of sight to WD0455-282 with three velocity components. To our knowledge, this is the first such detection of multi-component circumstellar absorption along the line of sight to a white dwarf., 19 pages, 23 figures, 8 tables. Accepted for publication in the Monthly Notices of the Royal Astronomical Society

We prove that the moduli space of compact genus three Riemann surfaces contains only finitely many algebraically primitive Teichmuller curves. For the stratum $\Omega\mathcal{M}_{3}(4)$ , consisting of holomorphic one-forms with a single zero, our approach to finiteness uses the Harder-Narasimhan filtration of the Hodge bundle over a Teichmuller curve to obtain new information on the locations of the zeros of eigenforms. By passing to the boundary of moduli space, this gives explicit constraints on the cusps of Teichmuller curves in terms of cross-ratios of six points on $\mathbf{P}^{1}$ . These constraints are akin to those that appear in Zilber and Pink’s conjectures on unlikely intersections in diophantine geometry. However, in our case one is lead naturally to the intersection of a surface with a family of codimension two algebraic subgroups of $\mathbf{G}_{m}^{n}\times\mathbf{G}_{a}^{n}$ (rather than the more standard $\mathbf{G}_{m}^{n}$ ). The ambient algebraic group lies outside the scope of Zilber’s Conjecture but we are nonetheless able to prove a sufficiently strong height bound. For the generic stratum $\Omega\mathcal{M}_{3}(1,1,1,1)$ , we obtain global torsion order bounds through a computer search for subtori of a codimension-two subvariety of $\mathbf{G}_{m}^{9}$ . These torsion bounds together with new bounds for the moduli of horizontal cylinders in terms of torsion orders yields finiteness in this stratum. The intermediate strata are handled with a mix of these techniques.

White dwarf atmospheres are subjected to gravitational potentials around $10^5$ times larger than occur on Earth. They provide a unique environment in which to search for any possible variation in fundamental physics in the presence of strong gravitational fields. However, a sufficiently strong magnetic field will alter absorption line profiles and introduce additional uncertainties in measurements of the fine structure constant. Estimating the magnetic field strength is thus essential in this context. Here we model the absorption profiles of a large number of atomic transitions in the white dwarf photosphere, including first-order Zeeman effects in the line profiles, varying the magnetic field as a free parameter. We apply the method to a high signal-to-noise, high-resolution, far-ultraviolet HST/STIS spectrum of the white dwarf G191-B2B. The method yields a sensitive upper limit on its magnetic field of $B < 2300$ Gauss at the $3\sigma$ level. Using this upper limit we find that the potential impact of quadratic Zeeman shifts on measurements of the fine structure constant in G191-B2B is 4 orders of magnitude below laboratory wavelength uncertainties., Comment: 10 pages, 5 figures

The presence of ultra-high excitation (UHE) absorption lines (e.g., O VIII) in the optical spectra of several of the hottest white dwarfs poses a decades-long mystery and is something that has never been observed in any other astrophysical object. The occurrence of such features requires a dense environment with temperatures near $10^6$K, by far exceeding the stellar effective temperature. Here we report the discovery of a new hot wind white dwarf, GALEXJ014636.8+323615. Astonishingly, we found for the first time rapid changes of the equivalent widths of the UHE features, which are correlated to the rotational period of the star ($P=0.242035$d). We explain this with the presence of a wind-fed circumstellar magnetosphere in which magnetically confined wind shocks heat up the material to the high temperatures required for the creation of the UHE lines. The photometric and spectroscopic variability of GALEXJ014636.8+323615 can then be understood as consequence of the obliquity of the magnetic axis with respect to the rotation axis of the white dwarf. This is the first time a wind-fed circumstellar magnetosphere around an apparently isolated white dwarf has been discovered and finally offers a plausible explanation of the ultra hot wind phenomenon., Published in MNRAS Letters

RIZ1 is a histone methyltransferase whose expression and activity are reduced in many cancers. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located and with decreased RIZ1 expression. Forced RIZ1 expression in model CML blast crisis (BC) cell lines decreases proliferation, increases apoptosis and enhances differentiation. We characterized molecular mechanisms that may contribute to potential CML tumor suppressor properties of RIZ1. Several RIZ1-regulated genes involved in insulin-like growth factor-1 (IGF-1) signaling were identified using cDNA microarrays. RIZ1 was shown to associate with promoter regions of IGF-1 and to increase histone H3 lysine 9 methylation using chromatin immunoprecipitation assays. IGF-1-blocking antibody was used to demonstrate the importance of autocrine IGF-1 signaling in CML-BC cell line viability. Forced RIZ1 expression in CML-BC cell lines decreases IGF-1 receptor activation and activation of downstream signaling components extracellular signal-regulated kinase 1/2 and AKT. These results highlight the therapeutic potential of inhibiting IGF-1 pathway in the acute phase of CML.

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies., Wellcome Trust (London, England) (Core Award 090532/Z/09/Z), Wellcome Trust (London, England) (Senior Investigator Award 095552/Z/11/Z ), Wellcome Trust (London, England) (WT095908), Wellcome Trust (London, England) (WT109497), Wellcome Trust (London, England) (WT098051), Wellcome Trust (London, England) (WT086084/Z/08/Z), Wellcome Trust (London, England) (WT100956/Z/13/Z ), Wellcome Trust (London, England) (WT097307), Wellcome Trust (London, England) (WT0855322/Z/08/Z ), Wellcome Trust (London, England) (WT090770/Z/09/Z ), Wellcome Trust (London, England) (Major Overseas program in Vietnam grant 089276/Z.09/Z), Medical Research Council (Great Britain) (grant G0801823), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I02593X/1), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I021213/1), Zhongguo ke xue ji shu qing bao yan jiu suo. Office of 863 Programme of China (2012AA02A201), National Basic Research Program of China (2011CB809201), National Basic Research Program of China (2011CB809202), National Basic Research Program of China (2011CB809203), National Natural Science Foundation of China (31161130357), Shenzhen Municipal Government of China (grant ZYC201105170397A), Canadian Institutes of Health Research (grant 136855), Quebec Ministry of Economic Development, Innovation, and Exports (PSR-SIIRI-195), Germany. Bundesministerium für Bildung und Forschung (0315428A), Germany. Bundesministerium für Bildung und Forschung (01GS08201), Germany. Bundesministerium für Bildung und Forschung (BMBF-EPITREAT grant 0316190A), Deutsche Forschungsgemeinschaft (Emmy Noether Grant KO4037/1-1), Beatriu de Pinos Program (2006 BP-A 10144), Beatriu de Pinos Program (2009 BP-B 00274), Spanish National Institute for Health (grant PRB2 IPT13/0001-ISCIII-SGEFI/FEDER), Japan Society for the Promotion of Science (fellowship number PE13075), Marie Curie Actions Career Integration (grant 303772), Fonds National Suisse del la Recherche, SNSF, Scientifique (31003A_130342), National Center for Biotechnology Information (U.S.) (U54HG3067), National Center for Biotechnology Information (U.S.) (U54HG3273), National Center for Biotechnology Information (U.S.) (U01HG5211), National Center for Biotechnology Information (U.S.) (U54HG3079), National Center for Biotechnology Information (U.S.) (R01HG2898), National Center for Biotechnology Information (U.S.) (R01HG2385), National Center for Biotechnology Information (U.S.) (RC2HG5552), National Center for Biotechnology Information (U.S.) (U01HG6513), National Center for Biotechnology Information (U.S.) (U01HG5214), National Center for Biotechnology Information (U.S.) (U01HG5715), National Center for Biotechnology Information (U.S.) (U01HG5718), National Center for Biotechnology Information (U.S.) (U01HG5728), National Center for Biotechnology Information (U.S.) (U41HG7635), National Center for Biotechnology Information (U.S.) (U41HG7497), National Center for Biotechnology Information (U.S.) (R01HG4960), National Center for Biotechnology Information (U.S.) (R01HG5701), National Center for Biotechnology Information (U.S.) (R01HG5214), National Center for Biotechnology Information (U.S.) (R01HG6855), National Center for Biotechnology Information (U.S.) (R01HG7068), National Center for Biotechnology Information (U.S.) (R01HG7644), National Center for Biotechnology Information (U.S.) (DP2OD6514), National Center for Biotechnology Information (U.S.) (DP5OD9154), National Center for Biotechnology Information (U.S.) (R01CA166661), National Center for Biotechnology Information (U.S.) (R01CA172652), National Center for Biotechnology Information (U.S.) (P01GM99568), National Center for Biotechnology Information (U.S.) (R01GM59290), National Center for Biotechnology Information (U.S.) (R01GM104390), National Center for Biotechnology Information (U.S.) (T32GM7790), National Center for Biotechnology Information (U.S.) (R01HL87699), National Center for Biotechnology Information (U.S.) (R01HL104608), National Center for Biotechnology Information (U.S.) (T32HL94284), National Center for Biotechnology Information (U.S.) (HHSN268201100040C), National Center for Biotechnology Information (U.S.) (HHSN272201000025C), Lundbeck Foundation (grant R170-2014-1039, Simons Foundation (SFARI award SF51), National Science Foundation (U.S.) (Research Fellowship DGE-1147470)

The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½-year old female patient with a 2-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.

During lytic infection in epithelial cells the expression of herpes simplex virus type 1 (HSV-1) immediate-early (IE) genes is initiated by a multiprotein complex comprising the virion-associated protein VP16 and two cellular proteins, host cellular factor (HCF) and Oct-1. Oct-1 directly recognizes TAATGARAT elements in promoters of IE genes. The role of HCF is not clear. HSV-1 also infects sensory neurons innervating the site of productive infection and establishes a latent infection in these cells. It is likely that some VP16 is retained by the HSV-1 nucleocapsid as it reaches the neuronal nucleus. Its activity must therefore be suppressed for successful establishment of viral latency. Recently, we discovered an HCF-binding cellular protein called Zhangfei. Zhangfei, in an HCF-dependent manner, inhibits Luman/LZIP/CREB3, another cellular HCF-binding transcription factor. Here we show that Zhangfei is selectively expressed in human neurons. When delivered to cultured cells that do not normally express the protein, Zhangfei inhibited the ability of VP16 to activate HSV-1 IE expression. The inhibition was specific for HCF-dependent transcriptional activation by VP16, since a Gal4-VP16 chimeric protein was inhibited only on a TAATGARAT-containing promoter and not a on a Gal4-containing promoter. Zhangfei associated with VP16 and inhibited formation of the VP16-HCF-Oct-1 complex on TAATGARAT motifs. Zhangfei also suppressed HSV-1-induced expression of several cellular genes including topoisomerase IIα, suggesting that in addition to suppressing IE expression Zhangfei may have an inhibitory effect on HSV-1 DNA replication and late gene expression.

Summary: Next-generation sequencing can provide insight into protein–DNA association events on a genome-wide scale, and is being applied in an increasing number of applications in genomics and meta-genomics research. However, few software applications are available for interpreting these experiments. We present here an efficient application for use with chromatin-immunoprecipitation (ChIP-Seq) experimental data that includes novel functionality for identifying areas of gene enrichment and transcription factor binding site locations, as well as for estimating DNA fragment size distributions in enriched areas. The FindPeaks application can generate UCSC compatible custom ‘WIG’ track files from aligned-read files for short-read sequencing technology. The software application can be executed on any platform capable of running a Java Runtime Environment. Memory requirements are proportional to the number of sequencing reads analyzed; typically 4 GB permits processing of up to 40 million reads. Availability: The FindPeaks 3.1 package and manual, containing algorithm descriptions, usage instructions and examples, are available at http://www.bcgsc.ca/platform/bioinfo/software/findpeaks Source files for FindPeaks 3.1 are available for academic use. Contact: afejes@bcgsc.ca

RIZ1 (PRDM2) is a tumor suppressor gene whose expression and activity are reduced by genetic and epigenetic aberrations in many cancers. In chronic myeloid leukemia (CML), blastic transformation is associated with loss of heterozygosity at 1p36, the region where RIZ1 is located, suggesting that RIZ1 has an essential role in CML pathogenesis. In CML patients and in the CML blast crisis cell line K562, we observed aberrant RIZ1 promoter methylation. To further characterize RIZ1 tumor suppressor properties that are related to CML, we analyzed RIZ1-induced changes in proliferation, apoptosis, and differentiation in CML myeloid blast crisis (CML-BC) cell lines (K562, ERY-1, YN-1, and JURL-MK1) that express low levels of endogenous RIZ1. Forced RIZ1 expression in CML-BC cell lines substantially decreased proliferation, increased apoptosis, and increased the population of cells in G2/M phase of the cell cycle. RIZ1 expression also promoted differentiation as assessed by benzidine staining in CML-BC cell lines expressing immature erythroid cell features (K562, ERY-1, YN-1) and by CD117 and CD33 expression in JURL-MK1, a CML-BC cell line expressing megakaryoblastic features. To identify genes and pathways influenced by RIZ1 expression, we used 42k cDNA microarrays to globally monitor how RIZ1 expression changes the gene expression profile of K562 cells. We discovered 25 RIZ1-regulated genes that are involved in a variety of biological processes with a subgroup of genes involved in IGF-1 signaling. The most strongly RIZ1 down-regulated genes (IGF1) and up-regulated genes (SPARC, IGFBP2) are involved in IGF-1 signaling. Using chromatin immunoprecipitation (ChIP), we determined that RIZ1 associates with IGF-1 and SPARC promoters. RIZ1 contains a PR domain that has Histone H3 lysine 9 methylation activity, which is implicated in gene repression. Using ChIP assays, we found that RIZ1 expression in K562 cells increased Histone H3 lysine 9 methylation of the IGF-1 promoter. The increased Histone H3 lysine 9 methylation is associated with decreased IGF-1 expression as monitored by RT-PCR and Western analysis. We observed autocrine production of IGF-1 in K562 cells, by culturing cells in serum free media and monitoring IGF-1 production and signaling. We detected receptor bound IGF-1 by flow cytometry, and compared the growth properties of sorted IGF-1 positive and negative cells. IGF-1 positive cells have increased number of cells in G2/M and S phase, a reduced number of cells in G1 and higher numbers of mitoses and Ki-67 positive nuclei compared with IGF-1 negative cells. RIZ1 expression in K562 decreased the amount of receptor bound IGF-1, reduced IGF-1 receptor activation, and reduced the activity of downstream IGF-1 signaling pathways. RIZ1 expression in K562 substantially reduced AKT1 and ERK1/2 phosphorylation. Our study demonstrates that RIZ1 reduces cell proliferation, increases apoptosis, and enhances differentiation of CML blast crisis cell lines. RIZ1 also controls autocrine production of IGF-1 and blocks the activity of IGF-1 signaling pathways. These activities may in part be responsible for RIZ1 tumor suppressor activity and point to the therapeutic potential of IGF-1 pathway inhibition in the acute phase of CML.

RIZ1 (PRDM2) is a tumor suppressor gene on 1p36 that frequently undergoes deletion, rearrangements, and loss of heterozygosity in a broad spectrum of tumors. RIZ1 is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 contains a ~130 amino acid conserved domain (PR or SET) that is important in chromatin-mediated regulation of gene expression and in the development of cancer. RIZ1 methylates Histone H3 on K9 and this activity may play a role in transcription repression as H3-K9 methylation is known to be associated with repression. Aberrant activities or mistargeting of chromatin modifying activities are proving to have unexpected links to cancer. We and others have shown that RIZ1 expression is down regulated in human leukemias and in the human erythroleukemia cell line K562. Expression of RIZ1 in K562 reduced proliferation, increased apoptosis, and promoted erythroid differentiation. To understand how RIZ1’s DNA binding, methyltransferase, and transcription repressor functions are related to its tumor suppressor activity it is necessary to characterize RIZ1 target genes. We used DNA microarrays to globally monitor how RIZ1 affects gene expression profiles. We constructed a K562 cell line with RIZ1 stably integrated under the control of a CMV promoter and analyzed the gene expression profiles of K652 and K562 + RIZ1 using a 42K Stanford human gene microarray. By comparing the gene expression profiles of these cell lines, we identified potential RIZ1 gene targets that are up and down regulated in the presence of RIZ1. In total, we identified 5 upregulated genes and 20 down regulated genes using significance analysis of microarrays (SAM) and standard deviation filter analysis of the gene expression data. RIZ1-mediated changes in gene expression profiling indicate that RIZ1 is potentially involved in the regulation and connection of the IGF-1 (IGF-1, IGFBP2) and integrin (LMS1) pathways, and in the activation of the TGF-β (SPARC) pathway. The genes perturbed by RIZ1 expression suggest that the tumor suppressor properties of RIZ1 arise from its control of proliferation, apoptosis and differentiation using these pathways. Finally, we observed an overrepresentation of the SP-1 transcription factor binding sites in genes that are upregulated in the absence of RIZ1. This correlates with the ability of RIZ1 to recognize SP1 sequences.

Gene promoter methylation is a major mechanism of transcriptional silencing in cancer cells that is mediated by CpG methylation and histone deacetylase activity, with methylation being dominant. Importantly, pharmacological treatment of cancer cells with inhibitors of DNA methylation and histone deacetylation have been shown to synergistically reactivate transcription of previously silenced genes. In an effort to identify novel genes silenced by DNA methylation in human acute myeloid leukemia (AML), we used cDNA microarray technology to screen for genes upregulated in the human cell line AML193 following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, Trichostatin A (TSA). Analysis of 1,700 gene microarrays in triplicate revealed seven genes consistently upregulated by combined 5-Aza-dC and TSA treatment, all of which were confirmed by semi-quantitative RT-PCR. Interestingly, four of these genes (MT1H, MT1E, MT2A, MT1G) are members of the metallothionein family of cysteine-rich small molecular weight proteins. These proteins are considered to be important mediators of cellular detoxification and are induced by toxic heavy metals, UV irradiation and reactive oxygen species. As well, MT1G promoter methylation has been implicated in the pathogenesis of sporadic papillary thyroid carcinoma. The methylation status of candidate metallothionein genes reactivated by 5-Aza-dC and TSA treatment in AML193 cells was assessed by methylation specific PCR (MSP) in seven human AML cell lines. Methylated promoter alleles were detected in MT1H, MT1E and MT1G in 71%, 29% and 29% of cell lines, respectively, and there was no methylation of MT2A in any of the cell lines tested. To investigate for the occurrence of MT1H, MT1E and MT1G methylation in human AML in vivo, we analyzed AML patient blasts (n=39) and non-leukemic controls (n=13) by MSP. MT1H, MT1E and MT1G methylation was detected in 51%, 0% and 3% of AML patient samples, respectively, and there was no methylation of any of these genes in control samples. Our findings implicate MT1H promoter methylation in the pathogenesis of human AML and suggest that the use of cDNA microarray technology following pharmacological manipulation is a useful approach for identifying novel epigenetically silenced genes in this disease.

Background Rare coding variants constitute an important class of human genetic variation, but are underrepresented in current databases that are based on small population samples. Recent studies show that variants altering amino acid sequence and protein function are enriched at low variant allele frequency, 2 to 5%, but because of insufficient sample size it is not clear if the same trend holds for rare variants below 1% allele frequency. Results The 1000 Genomes Exon Pilot Project has collected deep-coverage exon-capture data in roughly 1,000 human genes, for nearly 700 samples. Although medical whole-exome projects are currently afoot, this is still the deepest reported sampling of a large number of human genes with next-generation technologies. According to the goals of the 1000 Genomes Project, we created effective informatics pipelines to process and analyze the data, and discovered 12,758 exonic SNPs, 70% of them novel, and 74% below 1% allele frequency in the seven population samples we examined. Our analysis confirms that coding variants below 1% allele frequency show increased population-specificity and are enriched for functional variants. Conclusions This study represents a large step toward detecting and interpreting low frequency coding variation, clearly lays out technical steps for effective analysis of DNA capture data, and articulates functional and population properties of this important class of genetic variation.