The origin and evolution of mutations in acute myeloid leukemia.

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

The Impact of Primary Care Clinic and Family Physician Continuity on Patient Health Outcomes: A Retrospective Analysis From Alberta, Canada.

PURPOSE: Continuity of care is broadly associated with better patient health outcomes. The relative contributions of continuity with an individual physician and with a practice, however, have not generally been distinguished. This retrospective observational study examined the impact of continuity of care for patients seen at their main clinic but by different family physicians. METHODS: We analyzed linked health administrative data from 2015-2018 from Alberta, Canada to explore the association of physician and clinic continuity with rates of emergency department (ED) visits and hospitalizations across varying levels of patient complexity. Physician continuity was calculated using the known provider of care index and clinic continuity with an analogous measure. We developed zero-inflated negative binomial models to assess the association of each with all-cause ED visits and hospitalizations. RESULTS: High physician continuity was associated with lower ED use across all levels of patient complexity and with fewer hospitalizations for highly complex patients. Broadly, no (0%) clinic continuity was associated with increased use and complete (100%) clinic continuity with decreased use, with the largest effect seen for the most complex patients. Levels of clinic continuity between 1% and 50% were generally associated with slightly higher use, and levels of 51% to 99% with slightly lower use. CONCLUSIONS: The best health care outcomes (measured by ED visits and hospitalizations) are associated with consistently seeing one's own primary family physician or seeing a clinic partner when that physician is unavailable. The effect of partial clinic continuity appears complex and requires additional research. These results provide some reassurance for part-time and shared practices, and guidance for primary care workforce policy makers.

Implementation of a Physician-Patient Attachment Initiative in Alberta.

Attachment to a primary care physician (PCP) is a foundational component of the Patient's Medical Home. Yet how can attachment exist in a system that does not limit where patients seek care? This article describes a top-down approach with the ideologies of a bottom-up collaborative to address attachment within an Alberta primary care network. The steps taken to reduce the number of patients listed on multiple PCP panels from 27% to 4% will be described. Learnings from this initiative suggest that direct involvement with providers, coupled with engaged physician leadership, can create a local system of information delivery that supports the attachment of patients to their most responsible PCP.

DNMT3A mutations in acute myeloid leukemia.

BACKGROUND: The genetic alterations responsible for an adverse outcome in most patients with acute myeloid leukemia (AML) are unknown. METHODS: Using massively parallel DNA sequencing, we identified a somatic mutation in DNMT3A, encoding a DNA methyltransferase, in the genome of cells from a patient with AML with a normal karyotype. We sequenced the exons of DNMT3A in 280 additional patients with de novo AML to define recurring mutations. RESULTS: A total of 62 of 281 patients (22.1%) had mutations in DNMT3A that were predicted to affect translation. We identified 18 different missense mutations, the most common of which was predicted to affect amino acid R882 (in 37 patients). We also identified six frameshift, six nonsense, and three splice-site mutations and a 1.5-Mbp deletion encompassing DNMT3A. These mutations were highly enriched in the group of patients with an intermediate-risk cytogenetic profile (56 of 166 patients, or 33.7%) but were absent in all 79 patients with a favorable-risk cytogenetic profile (P<0.001 for both comparisons). The median overall survival among patients with DNMT3A mutations was significantly shorter than that among patients without such mutations (12.3 months vs. 41.1 months, P<0.001). DNMT3A mutations were associated with adverse outcomes among patients with an intermediate-risk cytogenetic profile or FLT3 mutations, regardless of age, and were independently associated with a poor outcome in Cox proportional-hazards analysis. CONCLUSIONS: DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile and are independently associated with a poor outcome. (Funded by the National Institutes of Health and others.).

Association between continuity and access in primary care: a retrospective cohort study.

BACKGROUND: Continuity of care is a tenet of primary care. Our objective was to explore the relation between a change in access to a primary care physician and continuity of care. METHODS: We conducted a retrospective cohort study among physicians in a primary care network in southwest Alberta who measured access consistently between 2009 and 2016. We used time to the third next available appointment as a measure of access to physicians. We calculated the provider and clinic continuity, discontinuity and emergency department use based on the physicians' own panels. Physicians who improved, worsened or maintained their level of access within a given year were assessed in multilevel models to determine the association with continuity of care at the physician and clinic levels and the emergency department. RESULTS: We analyzed data from 190 primary care physicians. Physicians with improved access increased provider continuity by 6.8% per year, reduced discontinuity by 2.1% per year, and decreased emergency department encounters by 78 visits per 1000 patients per year compared to physicians with stable access. Physicians with worsening access had a 6.2% decrease in provider continuity and an increased number of emergency department encounters (64 visits per 1000 panelled patients per year) compared to physicians with stable access. INTERPRETATION: Changes in access to primary care can affect whether patients seek care from their own physician, from another clinic or at the emergency department. Improving access by reducing the delay in obtaining an appointment with one's primary care physician may be one mechanism to improve continuity of care.

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein.

Viruses have been subjected to intense study because of their medical importance and because they can provide fundamental insights into normal and pathological cellular processes. Indeed, much of our knowledge about basic cellular biology and biochemistry was acquired through the study of viruses, and some of medicine's greatest triumphs and challenges involve viruses. Since viruses have evolved to exploit important cell processes, they can provide tools and approaches to manipulate cell function. The small transmembrane E5 protein of bovine papillomavirus type 1 transforms cells by a unique mechanism involving ligand-independent activation of the platelet-derived growth factor beta receptor. Experiments summarized in this review suggest that it may be possible to use the E5 protein as a model to design an entirely new class of small, modular transmembrane proteins with novel biological activities.

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor.

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.

Conserved locus-specific silencing functions of Schizosaccharomyces pombe sir2+.

In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor.

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.

Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells.

To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.

Packing contacts can mediate highly specific interactions between artificial transmembrane proteins and the PDGFbeta receptor.

We used proteins with randomized transmembrane (TM) domains to explore the role of hydrophobic amino acids in mediating specific interactions between transmembrane helices. The 44-aa bovine papillomavirus E5 protein, which binds to the TM domain of the PDGFbeta receptor (PDGFbetaR) was used as a scaffold to construct a library encoding small dimeric proteins with randomized, strictly hydrophobic TM domains, and proteins were selected that induced focus formation in mouse C127 cells by activating the PDGFbetaR. Analysis of these proteins identified a motif of two hydrophobic residues that, when inserted into a 17-residue polyleucine TM domain, generated a protein that activated the PDGFbetaR and transformed cells. In addition, we identified transforming proteins that activated the wild-type PDGFbetaR but did not activate a series of PDGFbetaR TM point mutants that were efficiently activated by the E5 protein, indicating that these proteins were more specific than the E5 protein. Our results implied that multiple van der Waals interactions distributed along the entire length of the TM domains were required for productive interaction between the PDGFbetaR and some small proteins lacking hydrophilic TM residues. Our results also suggested that excluding hydrophilic residues from small TM proteins and peptides is a strategy to increase the specificity of heteromeric TM helix-helix interactions.

Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia.

Although dementia of the Alzheimer's type (DAT) is the most common form of dementia, the severity of dementia is only weakly correlated with DAT pathology. In contrast, postmortem measurements of cholinergic function and membrane ethanolamine plasmalogen (PlsEtn) content in the cortex and hippocampus correlate with the severity of dementia in DAT. Currently, the largest risk factor for DAT is age. Because the synthesis of PlsEtn occurs via a single nonredundant peroxisomal pathway that has been shown to decrease with age and PlsEtn is decreased in the DAT brain, we investigated potential relationships between serum PlsEtn levels, dementia severity, and DAT pathology. In total, serum PlsEtn levels were measured in five independent population collections comprising >400 clinically demented and >350 nondemented subjects. Circulating PlsEtn levels were observed to be significantly decreased in serum from clinically and pathologically diagnosed DAT subjects at all stages of dementia, and the severity of this decrease correlated with the severity of dementia. Furthermore, a linear regression model predicted that serum PlsEtn levels decrease years before clinical symptoms. The putative roles that PlsEtn biochemistry play in the etiology of cholinergic degeneration, amyloid accumulation, and dementia are discussed.

Pathology-guided MR analysis of acute and chronic experimental allergic encephalomyelitis spinal cord lesions at 1.5T.

PURPOSE: To directly correlate spinal cord pathology of guinea pigs with experimental allergic encephalomyelitis (EAE) to the MRI data obtained at 1.5T. MATERIALS AND METHODS: Spinal cords from EAE animals were imaged in vivo with the following MRI sequences: T2-FSE, PD-FSE, fluid-attenuated inversion recovery (FLAIR)-FSE, T2-CSE, T1-CSE, T1-CSE + gadolinium-DTPA (Gd-DTPA), PD-CSE, and short-tau inversion recovery (STIR)-FSE. The spinal cords were removed and the lesions with specific pathological compositions were identified by histological analysis. Regions of interest (ROIs) were drawn on the corresponding MR images, and signal-to-noise ratios (SNRs) were measured for each MR sequence and compared with controls. RESULTS: The receiver operating characteristic (ROC) analysis of STIR-FSE and PD-CSE was able to differentiate tissue that contained cellular infiltrates with a high degree of accuracy. The SNRs of T2-FSE, STIR-FSE, T2-CSE, PD-CSE, and T1-CSE + Gd-DTPA were elevated in lesions that contained cellular infiltrates alone, whereas the SNRs of PD-CSE and T1-CSE + Gd-DTPA were reduced in demyelinated lesions that also contained inflammation. CONCLUSION: The SNR difference between the two lesion groups suggests that the combination of STIR-FSE, PD-CSE, and T1-CSE + Gd-DTPA sequences may be useful for differentiating inflammatory lesions containing demyelination from lesions with inflammation alone.

In vivo 4.0-T magnetic resonance investigation of spinal cord inflammation, demyelination, and axonal damage in chronic-progressive experimental allergic encephalomyelitis.

PURPOSE: To image and dissect the lumbar spinal cord of guinea pigs with chronic-progressive experimental allergic encephalomyelitis (CP-EAE) and directly correlate the pathology to the magnetic resonance (MR) image data obtained at 4 T and determine if these MR contrasts can accurately differentiate a specific type of pathology from control tissue. MATERIALS AND METHODS: The amount of inflammation, demyelination, and axonal pathology were quantified in the whole cord cross sections. The signal intensities (SIs) for 228 individual regions of interest (ROIs) (normal-appearing white matter (NAWM) and tissue containing inflammation with or without demyelination) were measured directly from the corresponding area on the MR images. RESULTS: Conventional MR contrast SIs and magnetization transfer ratio (MTR) were related to the degree of demyelination and presence of inflammation. MTR and proton density-weighted (PDw) SIs were both moderately related to axonal density. The SIs for NAWM and in lesions containing both cellular infiltrates and demyelination in all conventional MR contrast images were also increased, whereas the MTR was decreased when compared to control tissue. CONCLUSION: The SIs from the conventional MR contrasts and MTR at 4 T were sensitive to the presence of disease within CP-EAE spinal cord, but were not specific to the underlying pathology.

Initial sequencing and comparative analysis of the mouse genome.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.