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I was reading recently about prion disease and it caught my attention that a normal prion protein is coded n chromosome 20, therefore, in order for an infectious prion protein to attack, there must be already normal unsedimented prion proteins, does that mean the deletion of chromosome 20 in meiosis lead to immunity against prion disease, since the protein is not coded, that is if the human survives without chromosome 20
I see one report of a viable autosomal monosomy,
but in general, they are not survivable.
https://www.ncbi.nlm.nih.gov/pubmed/4086989 https://www.biology.iupui.edu/biocourses/N100/2k2humancsomaldisorders.html https://www.ncbi.nlm.nih.gov/pubmed/29164644
A better question would be if person with a deletion or natural knockout of that gene are susceptible to that prion disease. Those subjects likely exist in larger numbers, though it's likely that few have been exposed to any particular prion disease.
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Progress and problems in the biology, diagnostics, and therapeutics of prion diseases
Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland.
Address correspondence to: Adriano Aguzzi, Institut für Neuropathologie, UniversitätsSpital Zürich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland. Phone: 41-1-255-2107 Fax: 41-1-255-4402 E-mail: [email protected]
Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland.
Address correspondence to: Adriano Aguzzi, Institut für Neuropathologie, UniversitätsSpital Zürich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland. Phone: 41-1-255-2107 Fax: 41-1-255-4402 E-mail: [email protected]
Find articles by Heikenwalder, M. in: JCI | PubMed | Google Scholar
Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland.
Address correspondence to: Adriano Aguzzi, Institut für Neuropathologie, UniversitätsSpital Zürich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland. Phone: 41-1-255-2107 Fax: 41-1-255-4402 E-mail: [email protected]
The term “prion” was introduced by Stanley Prusiner in 1982 to describe the atypical infectious agent that causes transmissible spongiform encephalopathies, a group of infectious neurodegenerative diseases that include scrapie in sheep, Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids, and bovine spongiform encephalopathy in cattle. Over the past twenty years, the word “prion” has been taken to signify various subtly different concepts. In this article, we refer to the prion as the transmissible principle underlying prion diseases, without necessarily implying any specific biochemical or structural identity. When Prusiner started his seminal work, the study of transmissible spongiform encephalopathies was undertaken by only a handful of scientists. Since that time, the “mad cow” crisis has put prion diseases on the agenda of both politicians and the media. Significant progress has been made in prion disease research, and many aspects of prion pathogenesis are now understood. And yet the diagnostic procedures available for prion diseases are not nearly as sensitive as they ought to be, and no therapeutic intervention has been shown to reliably affect the course of the diseases. This article reviews recent progress in the areas of pathogenesis of, diagnostics of, and therapy for prion diseases and highlights some conspicuous problems that remain to be addressed in each of these fields.
Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are invariably fatal neurodegenerative disorders affecting a broad spectrum of host species and arise via genetic, infectious, or sporadic mechanisms (Table 1). In humans, prion diseases result from infectious modes of transmission (variant Creutzfeldt-Jakob disease [vCJD], iatrogenic CJD, Kuru) inherited modes of transmission in which there is nonconservative germ line mutation of the PRNP gene open reading frame (familial CJD, Gerstmann-Sträussler-Scheinker Syndrome, Fatal Familial Insomnia) ( 1 , 2 ) and modes of transmission that have as yet been neither determined nor understood (sporadic CJD [sCJD]). The clinical symptoms associated with each of the human prion disease forms vary dramatically ( 2 ).
Spectrum of prion diseases of humans and animals
Nomenclature applied to prion biology continues to be complex and confusing to nonspecialists. Here we utilize the term “prion” to denote the causative agent of prion diseases, without implying associated structural properties. We refer to the disease-associated prion protein (PrP Sc ), a disease-specific isoform of the host-encoded cellular prion protein (PrP C ), which accumulates in individuals affected with most forms of TSE (Figure 1) ( 3 ). While PrP Sc is classically defined as partially protease-resistant, aggregated PrP, it has recently been shown that PrP C may undergo disease-associated structural modifications that do not impart properties of inherent protease resistance ( 4 ). In light of this, it is advisable that PrP Sc be defined on the basis of disease-associated structural modifications rather than properties of protease resistance.
Models of PrP C to PrP Sc conversion. (A) The heterodimer model proposes that upon infection of an appropriate host cell, the incoming PrP Sc (orange) starts a catalytic cascade using PrP C (blue) or a partially unfolded intermediate arising from stochastic fluctuations in PrP C conformations as a substrate, converting it by a conformational change into a new β-sheet–rich protein. The newly formed PrP Sc (green-orange) will in turn convert new PrP C molecules. (B) The noncatalytic nucleated polymerization model proposes that the conformational change of PrP C into PrP Sc is thermodynamically controlled: the conversion of PrP C to PrP Sc is a reversible process but at equilibrium strongly favors the conformation of PrP C . Converted PrP Sc is established only when it adds onto a fibril-like seed or aggregate of PrP Sc . Once a seed is present, further monomer addition is accelerated.
Prion diseases are conceptually recent the first cases of Creutzfeldt-Jakob disease were described eight decades ago ( 5 , 6 ), yet the protein-only theory of prion infection was originally formulated in 1967 ( 7 ) and later refined and the term “prion” coined in 1982 ( 8 ). The precise physical nature of the prion agent is still the subject of intense scientific controversy. PrP Sc may or may not be congruent with the infectious agent. It remains to be formally proven whether the infectious unit consists primarily or exclusively of: (a) a subspecies of PrP Sc (b) an intermediate form of PrP (PrP*) ( 9 ) (c) other host-derived proteins ( 10 ) or (d) nonprotein compounds (which may include glycosaminoglycans and maybe even nucleic acids) ( 11 ). We still do not know, therefore, whether the prion hypothesis is correct in its entirety.
As with any other disease, a thorough mechanistic understanding of pathogenesis is the best foundation for devising sensitive predictive diagnostics and efficacious therapeutic regimens.
The purpose of the present article is to discuss some aspects of the state of the art in prion science and their impact on prion diagnostics, primarily with respect to peripherally acquired prion disease. As of now, no causal therapies can be offered to prion disease victims. Yet we are witnessing the emergence of an impressive wealth of therapeutic approaches, some of which certainly deserve to be tested for their validity.
Prion pathogenesis is a dynamic process that can be broken down into spatially and temporally distinct phases: (a) infection and peripheral replication (b) transmigration from the periphery to the CNS (also termed “neuroinvasion”) and (c) neurodegeneration. But what are the mechanisms underlying neuroinvasion, and which cellular compartments are involved in replication and neuroinvasion of prions?
Cell tropism of prions varies dramatically among animal species and is also in part dependent on the particular strain of prion agent. For example, prions are lymphotropic in sheep scrapie and vCJD ( 12 ) but less so in sCJD ( 13 ) and bovine spongiform encephalopathy (BSE). Different prion strains can lead to different routes of peripheral replication in experimental models of scrapie ( 14 , 15 ), and, therefore, strain-encoded properties might also determine the route of peripheral replication. With respect to peripheral pathogenesis of prion diseases, it is well established that replication of the prion agent occurs in high titers in lymphoid tissues such as spleen and lymph nodes well before neuroinvasion and subsequent detection in the CNS ( 16 ).
Upon oral challenge, an early rise in prion infectivity is observed in the distal ileum of infected organisms. This applies to several species but has been most extensively investigated in sheep, and Western blot analyses and bioassays have shown that Peyer’s patches accumulate PrP Sc and contain high titers of prion infectivity. This is true also in the mouse model of scrapie, where administration of mouse-adapted scrapie prions (Rocky Mountain Laboratory [RML] strain) induces a surge in intestinal prion infectivity as early as a few days after inoculation ( 17 , 18 ). Indeed, immune cells are crucially involved in the process of neuroinvasion after oral application: mature follicular dendritic cells (FDCs), located in Peyer’s patches, may be critical for the transmission of scrapie from the gastrointestinal tract ( 16 , 18 ).
The resistance to prions of mice that lack expression of PrP C , encoded by Prnp (a single-copy gene located on chromosome 2 in mice and 20 in humans), is well documented ( 19 , 20 ). While the precise physiological function of PrP C is unclear, expression of it is absolutely required for transportation of the infectious agent both from the peripheral sites to the CNS ( 21 ) and within the CNS ( 22 ). However, reconstitution of Prnp knockout (Prnp o/o ) mice with WT bone marrow is insufficient to restore neuroinvasion in Prnp o/o mice ( 21 ). Hence one could argue that the elemental compartment required for prion neuroinvasion is stromal and must express PrP C . Nevertheless, in adoptive transfer experiments on Prnp o/o mice with WT bone marrow, the capability of the spleen to accumulate prions of the RML strain is restored ( 21 , 23 ). This suggests that hematopoietic cells transport prions from the entry site to the lymphoreticular system (LRS), which accumulates and replicates prions. B lymphocytes (not necessarily expressing PrP C ) are crucial for peripheral prion spread and neuroinvasion ( 24 – 26 ).
This dependence of FDCs on lymphotoxin (LT) signaling by B cells likely may explain — at least in part — the apparent requirement for B cells in peripheral pathogenesis: FDCs have been reported to accumulate PrP Sc following scrapie infection ( 27 ). Indeed, blockade of LT signaling via administration of a soluble, dimeric LTβ receptor immunoglobulin fusion protein (LTβR-Ig) ablates mature FDCs and significantly impairs peripheral prion pathogenesis ( 28 , 29 ).
FDCs are bifunctional cells: they support the formation and maintenance of the lymphoid microarchitecture but also play a role in antigen trapping — capturing immune complexes by Fcγ receptors and binding opsonized antigens to the CD21/CD35 complement receptors. Two studies have demonstrated that the complement system is relevant to prion pathogenesis. Mice genetically engineered to lack complement factors ( 30 ) or mice depleted of the C3 complement component ( 31 ) exhibited enhanced resistance to peripheral prion inoculation. However, FDCs are most likely immobile cells and therefore unlikely to be responsible for prion transport into the CNS.
But just which cell types are involved in neuroinvasion? The innervation pattern of lymphoid organs is primarily sympathetic ( 32 ). Sympathectomy delays the onset of scrapie, while sympathetic hyperinnervation enhances splenic prion replication and neuroinvasion, which suggests that innervation of secondary lymphoid organs is the rate-limiting step to neuroinvasion ( 33 ). However, there is no physical synapse between FDCs and sympathetic nerve endings ( 34 ). So how can prions transmigrate from FDCs to sympathetic nerve fibers? A series of recent experiments (discussed below) may go some way toward providing answers.
We investigated how the distance between FDCs and splenic nerves affects the velocity of neuroinvasion, utilizing mice deficient in the CXC chemokine receptor 5 (CXCR5), which directs lymphocytes toward specific microcompartments ( 35 ). While density, distribution, and branching patterns of sympathetic nerve processes in CXCR5 –/– spleens are normal, the distance between FDCs and nerve endings is greatly reduced ( 36 ).
After peripheral administration of high doses of prions, velocity of pathogenesis was similar in CXCR5 –/– and WT mice however, delivery of smaller inocula resulted in a dose-dependent increase in incubation periods in WT mice that was not evident in CXCR5 –/– mice. Peripheral prion pathogenesis in CXCR5 –/– mice is therefore more efficient upon incremental reduction of the inoculum.
What is the basis of this reduced incubation period? Kinetics measurements of prion infectivity titers in the thoracic spinal cord provided the answer: following peripheral administration, only traces of infectivity were found in WT spinal cords at 80 days post-inoculation (dpi), whereas infectivity rose to measurable levels in the spinal cords of CXCR5 –/– mice already at 60 dpi. This suggests that increased velocity of prion entry into the CNS in CXCR5 –/– mice is due to the repositioning of FDCs near highly innervated, splenic arterioles (Figure 2). This was validated by the finding that incubation periods were prolonged in CXCR5 –/– mice treated with soluble LTβR-Ig to deplete mature FDCs.
Positioning of FDCs in spleens of WT and CXCR5 –/– mice. (A and B) Diagrammatic representation of white pulp follicles in prion-infected CXCR5 –/– and WT mice. Anti-CD21 immunostaining was performed to visualize the lymphoid white pulp follicle microarchitecture. (C) Atypically localized perivascular FDCs in lymph follicles in CXCR5 –/– mice. Sympathetic nerves, visualized with antibodies to tyrosine hydroxylase (TH), are in close vicinity to FDCs (visualized by FDC-M1 immunostaining) (D). Scale bar: 50 mm. In contrast, sympathetic nerves in WT FDCs are localized in B cell areas at the periphery of the follicles (E and F). Arrowheads indicate TH and FDC-M1 positive areas. (G) Sympathetic nerves lining the thoracic spinal cord connect lymphoid organs and the CNS. (H) Shortened prion disease incubation period in CXCR5 –/– mice inoculated intraperitoneally, relative to WT controls.
Hence topographical relationships within lymphoid organs contribute to prion neuroinvasion. However, it remains to be determined whether this results from passive diffusion of prions or whether mobile cells (e.g., germinal center B cells) are involved in an active transport process.
This study also raises the possibility that spread of infection to peripheral nerves occurs more rapidly when FDCs are in close proximity to nerves in lymphoid tissue other than spleen, such as Peyer’s patches. Indeed, FDCs are crucial to disease progression but only during a short window of time following oral scrapie challenge ( 17 ). This implicates the efficiency of neuroimmune transfer of prions as a primary determinant of neuroinvasion. The detection of PrP Sc in spleens of sCJD patients ( 12 ) suggests that the interface between cells of the immune system and peripheral nerves (the neuroimmune connection) might also be of relevance in sporadic prion disease.
There has certainly been progress in understanding the events underlying peripheral prion pathogenesis and neuroinvasion ( 37 ). However, prions exert their destructive effects exclusively within the CNS. The precise cause of neurodegeneration remains poorly understood and is a point of contention among prionologists. It seems unlikely that PrP Sc is directly toxic, since tissue devoid of PrP C that subsequently accumulates PrP Sc remains healthy and free of pathology ( 20 , 38 ). During the conversion process, PrP C levels may be depleted, yet this is also an unlikely cause of pathology, since ablation of PrP C does not result in scrapie-like symptoms ( 39 ), even when ablated postnatally ( 40 ).
Lindquist and colleagues have suggested a mechanism that may account for prion-associated toxicity: (a) expression of a PrP variant resident in the cytosol was strongly neurotoxic in cultured cells and transgenic mice, which suggests a common framework for diverse PrP neurodegenerative disorders ( 41 ) and (b) PrP, retrogradely transported out of the endoplasmic reticulum, produced amorphous aggregates of PrP possessing partial proteinase K resistance in the cytosol. Once conversion occurred, it was self-sustaining ( 42 ). It will be interesting to determine whether the disease generated in these mice is, in some way, transmissible. However, while the results obtained here are certainly intriguing, it should be noted that reports elsewhere, although not refuting these observations, argue against the contribution of such potential neurotoxic PrP species ( 43 , 44 ). Similarly, it has been reported that PrP C in some forms of prion disease assumes a transmembrane topology, C-terminal transmembrane PrP ( Ctm PrP), and that the extent of neurotoxicity is a result of concentration of Ctm PrP, thereby arguing that Ctm PrP may represent a major toxic moiety ( 45 , 46 ). However, while we still do not understand the biochemical events involved in cytosolic or Ctm PrP-induced neurotoxicity, elucidation of this may aid in the much-needed identification of therapeutic targets. Additionally, in-depth characterization of transgenic mice expressing amino-terminally truncated PrP C ( 47 ), in which cerebellar neurodegeneration occurs, may not only aid in the elucidation of the molecular events responsible for potentially common neurodegenerative processes but perhaps also provide clues to the physiological function of PrP C itself.
The ability to secure early diagnosis is vital for therapeutic interventions to be of real value. With respect to animals destined for the human food chain, there is the additional demand to determine presence of the prion agent in tissues in asymptomatic organisms, well before the appearance of any clinical symptoms. This applies equally to the detection of prions in humans, who may participate in tissue donation programs.
Prions were transmitted via blood transfusion in sheep using blood obtained from infected animals prior to the onset of clinical symptoms ( 48 , 49 ). If the same route applies to humans, this could represent a nightmare scenario for the blood transfusion services ( 50 ). A transfusion recipient received blood from an individual harboring the vCJD agent 3.5 years prior to the development of any clinical signs of prion disease in the donor. The unfortunate recipient developed disease 6.5 years after the transfusion.
To be truly useful, prion diagnostics should identify “suspect” cases as early during pathogenesis as possible. However, the currently available methods are quite insensitive when compared with those available for other infectious diseases. PrP Sc represents the only disease-specific macromolecule identified to date, and all approved commercial testing procedures are based on the immunological detection of PrP Sc . While around 50 companies are reported to be developing prion diagnostic assays, all commercial test kits validated for use by the European Union rely on proteolytic removal of endogenous PrP C prior to detection of PrP Sc (Table 2). In addition, the conformation-dependent immunoassay ( 4 ) utilizes the differential binding of antibodies to native or denatured PrP Sc .
Molecular diagnosis of prion disease and prion infectivity
Circumvention of the protease digestion step might theoretically yield increased sensitivity of PrP Sc -based detection methods and make these methods more amenable to high-throughput technologies. However, it has proved difficult to discriminate between PrP C and PrP Sc with antibodies, despite some early reports ( 51 ). Interestingly, tyrosine-tyrosine-arginine (YYR) motifs ( 52 ) were reasoned to be more solvent-accessible in the pathological isoform of PrP, and a monoclonal antibody directed against these motifs was reported to be capable of selectively detecting PrP Sc across a variety of platforms. However, YYR motifs are certainly not unique to pathological prion proteins, and it remains to be determined whether this reagent can really improve the sensitivity of detection of prion infections.
Deposition of PrP Sc in lymphoid tissues of human prion disease patients has long been believed to be restricted to vCJD. However, recent results ( 12 ) imply that PrP Sc is present in spleens and muscle tissue from as much as one third of patients with sCJD. It is presently unclear whether the patients with extraneural PrP represent a specific subset of CJD patients or whether the extraneural-negative patients may simply deposit PrP Sc in muscle and spleen at levels that are below the detectability threshold of the assay. If the latter scenario proves true, and if the assay sensitivity can be raised, minimally invasive muscle biopsies may replace brain biopsy in clinical CJD diagnostics.
While presence of PrP Sc secures diagnostic association with the presence of prion disease, PrP Sc is not always easily detectable in several forms of prion disease ( 53 – 55 ). In order to enhance the safety of the blood supply and of products of bovine origin, absolute specificity in securing diagnosis of asymptomatic prion disease may not be required. Instead, it may be prudent to accommodate less than 100% specificity with a panel of surrogate markers capable of identifying donated blood units from “suspect” individuals rather than requiring definitive diagnosis. It could be envisaged that wide-scale primary screens accommodate a certain degree of loss of specificity to identify samples to be re-tested in a secondary screen utilizing more specific (and likely labor-intensive) criteria.
Several research efforts have been directed at identifying transcripts and proteins differentially expressed in tissues of prion-infected animals relative to disease-free controls ( 56 – 58 ). However, these have mostly focused primarily either on prion-infected neural cell lines or on CNS tissue, frequently with emphasis on late-stage disease. Ideally, these surrogate markers should be detectable (and differentially expressed) in easily accessible body fluids, such as blood or urine. At present, only one extraneural gene was reported to be differentially expressed during prion infection ( 59 ): erythroid differentiation–related factor (EDRF also known as erythroid-associated factor) levels were progressively reduced in spleens of prion-infected mice throughout pathogenesis and also in blood of experimentally infected mice, cattle with BSE, and sheep with clinically manifest scrapie.
Assessment of the levels of surrogate markers in healthy individuals is crucial in order to define the normal range of expression (according to age, sex, etc.) in order to determine what represents abnormal levels. In this respect, it is worth noting that determination of normal expression range must utilize appropriate controls. For example, EDRF transcript levels have recently been reported to show a broad range of variation in healthy human subjects ( 60 ). However, since EDRF is an erythroid-specific transcript, it would be imperative to utilize other erythroid transcripts as internal controls to normalize for variations in numbers of circulating cells in which the transcript under study is expressed relative to total cells. More searches for surrogate markers would certainly be useful, and it is likely that surrogate markers of prion disease, particularly if they are detectable in body fluids, will expand the panel of tools available for screening for prion infections.
It is also worth noting here that recent advances in neuroimaging techniques, particularly with respect to MRI, may lead to clinically useful methods of assessment of prion disease in humans, perhaps even the ability to distinguish between sCJD and vCJD ( 61 ). For example, in vCJD the pulvinar sign (a high T2 MRI signal in the posterior thalamus) has been suggested to be relatively specific for vCJD, being present in approximately 75% of vCJD patients tested ( 62 ). In sCJD, fluid-attenuated inversion recovery and diffusion-weighted MRI sequences appear to be associated with high sensitivity and specificity. MRI imaging techniques such as these may therefore represent a relatively noninvasive method to corroborate suspicion of clinical presentation of human prion disease.
While surrogate markers such as S-100, neuron-specific enolase, and 14-3-3 protein have been suggested as potential biomarkers of prion disease using body fluids such as cerebrospinal fluid (CSF) ( 63 , 64 ), it is worth remembering that these are clearly surrogate markers of general neurodegenerative disease and are not therefore predictive for human prion disease. For example, one study reported false positives of 14-3-3 detection in CSF samples of patients with herpes simplex encephalitis, hypoxic brain damage, atypical encephalitis, intracerebral metastases of a bronchial carcinoma, and metabolic encephalopathy ( 65 ).
It should not be forgotten that there is no ultimate consensus on the nature of the prion: PrP Sc itself might represent a surrogate marker of prion disease ( 66 ). The real gold standard of prion diagnostics is the detection of prion infectivity (whether or not PrP Sc is present). Until recently, the only method available to assay for prion infectivity was the use of the mouse bioassay, in which serial dilutions of test material are inoculated into mice and onset of disease noted. However, this procedure suffers from inaccuracy and is limited by the requirements for scores of mice and significant lengths of time. Recently, the use of highly susceptible cloned neural cell lines has provided what appears to be an assay that delivers a substantial reduction in both cost and time required to perform prion bioassays and may lend itself to high-throughput automation ( 67 ). Such assays may advance methodologies aimed at diagnostic assessment of the presence of the prion agent. However, it should be noted that these cell lines are currently reported only to be permissive to murine prions. It is to be expected that the spectrum of prion strains that can be assayed using this technology will expand.
For all the promising approaches that are being explored (Table 3), no therapy for prion diseases is available as of yet. Many substances appear to possess prion-curing properties in vitro, including Congo red ( 68 ), amphotericin B, anthracyclines ( 69 ), sulfated polyanions ( 70 ), porphyrins ( 71 ), branched polyamines ( 72 ), β-sheet breakers ( 73 ), the spice curcumin ( 74 ), and recently even small interfering RNAs ( 75 ). The majority of these molecules exert their biological effects by directly or indirectly interfering with conversion of PrP C to PrP Sc , thereby also aiding clearance of PrP Sc . Yet none of these compounds have proved very effective for actual therapy.
Approaches to prion therapy
In a recent report, results obtained in mice have led to the theory that administration of cytidyl-guanyl oligodeoxynucleotides (CpG-ODNs), which stimulate the innate immune system via toll-like receptor 9 (TLR9) signaling receptors on a variety of immune cells, may represent an applicable treatment regimen to delay prion disease in humans ( 76 ). Here it was shown that the incubation period of prion disease was extended in mice multidose treated with CpG-ODNs for twenty days. It was concluded that stimulation of innate immunity accounts for this apparent anti-prion effect, possibly through induction of anti-PrP antibodies. However, this is difficult to reconcile with several studies indicating that immune deficiencies of various sorts inhibit prion pathogenesis ( 24 , 25 , 30 , 77 ). In addition, prion pathogenesis is unhampered in MyD88 –/– mice, in which there is impaired TLR9 signaling ( 78 ). In fact, repeated CpG-ODN treatment has severe side effects, ranging from lymphoid follicle destruction and impaired antibody class switch to the development of ascites, hepatotoxicity, and thrombocytopenia ( 79 ). In addition, anti-PrP antibodies are not detectable in CpG-ODN–treated mice ( 79 ). It is likely therefore that the anti-prion effects of repeated CpG-ODN treatment arise via indiscriminate and undesirable follicular destruction.
Anti-PrP antibodies ( 30 ) and F(ab) fragments to PrP ( 80 , 81 ) can suppress prion replication in cultured cells. However, the mammalian immune system is essentially tolerant to PrP C ( 82 ). Ablation of Prnp ( 39 ) renders mice highly susceptible to immunization with prions ( 22 ). Tolerance can be circumvented by transgenic expression of an immunoglobulin μ chain containing the epitope-interacting region of a high-affinity anti-PrP monoclonal antibody. This sufficed to block prion pathogenesis upon intraperitoneal prion inoculation ( 83 ). Passive immunization may be a useful strategy for prophylaxis of prion diseases, since it has been shown that passive transfer of anti-PrP monoclonal antibodies prior to the onset of clinical symptoms is able to delay the onset of prion disease in mice inoculated intraperitoneally ( 84 ). Unfortunately, several efforts aimed at active immunization strategies have met with little success due to the robust immune tolerance to PrP C . In this respect, it is certainly worth noting that extensive neuronal apoptosis in hippocampus and cerebellum has been shown following intracranial delivery of monoclonal antibodies reactive against a subset of PrP epitopes ( 85 ). The implications here are obvious clearly, exhaustive in-vivo safety trials must be performed prior to the utilization of such strategies in humans.
Do any serious candidates for prion therapeutic strategies exist? It is well established that expression of two PrP C moieties that differ subtly from each other are able to inhibit prion replication ( 10 ). For example, humans heterozygous for a common PRNP polymorphism at codon 129 are largely protected from CJD ( 86 ). Although the precise molecular basis for this effect is unclear, it is possible that heterologous PrP C may exert inhibitory action on prion replication by sequestration. This has been addressed directly by fusion of PrP C to an immunoglobulin Fcγ domain ( 87 ), allowing for ligand dimerization, expression of the molecule as a soluble moiety, and also, therefore, increased overall stability in body fluids. Transgenetic expression of this PrP-Fc2 fusion protein resulted in significant prolongation of incubation period upon prion inoculation via competition with PrP Sc . It remains to be established whether PrP-Fc2 is acting as an anti-prion compound when delivered exogenously. If so, soluble prion protein mutants may well represent anti-prion compounds.
Prion diseases continue to present a diagnostic and therapeutic challenge to clinicians and researchers worldwide. There are many aspects of prion biology that remain unclear we still do not know the precise physical nature of the infectious agent, the molecular and biochemical mechanisms underlying associated neurodegeneration, or the physiological function of PrP C . The diagnostic tools currently available for prion diseases are significantly less sensitive and satisfactory than those available for other infectious diseases. Additionally, there is a dearth of therapeutic intervention strategies available for these diseases. However, that said, the last decade or so of prion research has witnessed astounding advances in our knowledge and understanding of basic prion biology, and the field has attracted increasing numbers of researchers from diverse disciplines. Undoubtedly, this trend will trigger further important advances in prion science.
Nonstandard abbreviations used: bovine spongiform encephalopathy (BSE) cellular prion protein (PrP C ) Creutzfeldt-Jakob disease (CJD) C-terminal transmembrane PrP ( Ctm PrP) CXC chemokine receptor 5 (CXCR5) cytidyl-guanyl oligodeoxynucleotide (CpG-ODN) days post-inoculation (dpi) disease-associated prion protein (PrP Sc ) erythroid differentiation–related factor (EDRF) follicular dendritic cell (FDC) LTβ receptor immunoglobulin fusion protein (LTβR-Ig) lymphoreticular system (LRS) lymphotoxin (LT) Prnp knockout (Prnp o/o ) Rocky Mountain Laboratory (RML) sporadic Creutzfeldt-Jakob disease (sCJD) toll-like receptor 9 (TLR9) transmissible spongiform encephalopathy (TSE) tyrosine-tyrosine-arginine (YYR) variant Creutzfeldt-Jakob disease (vCJD).
Conflict of interest: The authors have declared that no conflict of interest exists.
In this study, we used Fab phage display to retrieve antibodies targeting different epitopes of PrP from a synthetic human antibody library. We demonstrated that antibodies targeting the N-terminal part of PrP were neuroprotective in a model of prion-induced neurodegeneration. Interestingly, mining of published human antibody databases confirmed the presence of such anti-PrP antibodies in naïve repertoires of circulating B cells from healthy humans. We also found high-titer PrP autoantibodies directed against the flexible tail of PrP in the plasma of unselected hospitalized patients without any clinical features of a pathological disease.
Our data demonstrate the presence of naturally occurring, innocuous anti-PrP antibodies in humans which may constitute a potential source for the development of effective and safe immunotherapeutics to combat prion diseases.
Identification of Avr1
In an initial analysis of the xylem sap proteome of tomato plants infected with Fol race 1 using 2-D gel electrophoresis and mass spectrometry, three small secreted proteins of Fol were identified in addition to Avr3 (Six1), named Six2, Six3 and Six4, and their genes cloned . We now find that one of these, Six4, is not secreted by Fol race 2 (Fig. 1). For reasons detailed below, we now call this protein Avr1. Like the AVR3 (SIX1) gene, AVR1 is surrounded by repetitive elements (Fig. 2A). In all of the race 1 strains we examined, PCR experiments detected the presence of AVR1 and no sequence polymorphism was detected in the coding regions of seven isolates from different clonal lines (see  for the list of strains 17 of these are race 1, 23 are race 2 or 3). AVR1 was not detected in race 2 or 3 strains by PCR nor is AVR1 present in the genome sequence of the race 2 strain 4287 (Fusarium oxysporum Sequencing Project Broad Institute of Harvard and MIT (http://www.broad.mit.edu)). Absence of AVR1 or closely related genes in the race 2 and race 3 strains used in this study was confirmed by DNA gel blot analysis (Fig. 2B, lanes 4 and 7, respectively).
Proteins present in xylem sap of susceptible tomato plants infected with race 1 strain Fol004 (left panel) or race 2 strain Fol002 (right panel) were isolated and separated with 2-dimensional gel electrophoresis. Positions of isoelectric point markers are indicated at the top positions of molecular weight markers are indicated on the left. The arrows in the left panel point to the two spots previously shown to contain Avr1 (Six4)  the arrows in the right panel point to the corresponding (empty) positions. The right spot in the left panel likely represents a more extensively N-terminally processed form of Avr1 .
A) The AVR1 open reading frame (ORF open arrow) is interrupted by a single intron (black box)  (accession AM234064). The ORF is flanked 714 bp upstream by a copy of the transposon Tfo1 (striped arrow represents the end of the transposase ORF triangle represents the inverted repeat), 485 bp upstream by a partial miniature impala repetitive element (mimp-Δ, grey box triangle represents inverted repeat) and downstream by a Fot5-like repetitive element (the transposase ORF ends 541 bp downstream of the AVR1 ORF and is shown as a grey arrow). The small arrows denote the primers used to construct an AVR1 disruption construct and an AVR1 expression cassette for transformation to Fol (see Materials and methods). The insertion of a hygromycine resistance (hygR) cassette to create an AVR1 knock-out mutant is shown (not drawn to scale). The position of the probe and the restriction sites used for Southern blot analysis are indicated H: HindIII, B: BamHI. B) Southern blot confirming AVR1 disruption and ectopic insertion of AVR1. A Southern blot of genomic DNA digested with HindIII and BamHI was probed with a 1.4 kb probe encompassing the AVR1 ORF and 3′ sequences as indicated in Fig. 2A. The AVR1 locus in race 1 strain Fol004 (lane 1) is visible as a 1.25 kb HindIII band containing the ORF (AVR1) and a band of ∼5 kb containing sequences 3′ of the ORF (3′). In the race 1 avr1Δ strain (lane 2), replacement of the ORF with the disruption cassette through homologous recombination led to the expected replacement of the 1.25 HindIII band with a 1.1 kb BamHI-HindIII band containing part of the ORF and part of the disruption cassette (avr1Δ). Transformation of the AVR1 expression cassette to the avr1Δ strain (lane 3) led to reappearance of the AVR1 band. Race 2 strain Fol007 (lane 4) and race 3 strain Fol029 (lane 7) do not contain AVR1 (the AVR1 and 3′ bands are absent). Transformation of the AVR1 expression cassette to these strains (lanes 5 and 6: race 2 transformants lanes 8 and 9: race 3 transformants) leads to appearance of the 1.25 kb HindIII AVR1 band as well as a 0.56 kb HindIII-BamHI band (3′ ectopic) that comprises sequences 3′ of the AVR1 ORF until the BamHI site at the 3′ end of the expression cassette (which is not present in the genomic locus but corresponds to the end of the probe shown in Fig. 2A). Note that in the avr1Δ strain (lane 2) the 0.56 kb band indicative of ectopic insertion is also present, indicating that this strain contains an additional copy of the disruption cassette. The additional, weaker bands are probably due to 104 bp of non-coding sequence of the Fot5-like transposon present at the 3′ end of the probe (thick line next to the grey arrow in Fig. 2A) – there are seven copies of this sequence in the latest release of the genome sequence of race 2 strain 4287 (Fusarium oxysporum Sequencing Project Broad Institute of Harvard and MIT (http://www.broad.mit.edu). Molecular weight markers are indicated on the left (in kb).
To test whether AVR1 is indeed responsible for avirulence of Fol on plants carrying the I gene, we created an AVR1 gene knock-out in a race 1 strain (Fol004) through Agrobacterium-mediated transformation (Fig. 2). For the AVR1 gene, the frequency of homologous recombination leading to gene knock-out turned out to be extremely low, with only a single knock-out mutant obtained out of ∼200 transformants (Fig. 2B, lane 2). A disease assay with this mutant (avr1Δ) confirmed that indeed deletion of AVR1 leads to breaking of I-mediated disease resistance (Fig. 3A, panel A, quantified in Fig. 3B). Re-introduction of AVR1 in the avr1Δ strain (Fig. 2B, lane 3) restored the original avirulence phenotype (results not shown). In addition, we found that disease resistance conferred by the unlinked I-1 gene in tomato also depends on recognition of Avr1, since the avr1Δ strain (but not its parental strain) is virulent on a plant line carrying I-1 (line 90E402F, results not shown). This suggests that I and I-1 express the same resistance specificity.
Ten day old seedlings of tomato were inoculated with a fungal spore suspension and disease was scored after three weeks. Tomato lines carrying only a single resistance gene or no resistance gene were used to determine the effect of Avr1 on the activity of each resistance gene (see Materials and methods for description of plant lines). All lines were inoculated with the following Fol strains: race 1 (strain Fol004), race 2 (strain Fol007), race 3 (strain Fol029), race 1 avr1Δ (Fol004 with AVR1 deleted by gene replacement), race 2+AVR1 (Fol007 transformed with AVR1 similar virulence patterns were obtained with six independent transformants ) and race 3+AVR1 (Fol029 transformed with AVR1 similar virulence patterns were obtained with four independent transformants). A) Representative plants are shown three weeks after infection. Panel A shows that loss of AVR1 leads to breaking of I-mediated resistance. Panel B and C show that gain of AVR1 triggers I-mediated resistance. Panel D shows that loss of AVR1 leads to loss of virulence on I-2 and I-3-containing plant lines. Panels E and F show that gain of AVR1 by race 2 or race 3 leads to virulence on I-2 and I-3-containing plant lines. B): Quantification of disease assays. The outcomes of the disease assays depicted in (A) were quantified in two ways: 1) average plant weight above the cotyledons and 2) phenotype scoring according to a disease index ranging from zero (no disease) to four (heavily diseased or dead). Error bars indicate the 95% confidence interval of the mean. Interactions where Avr1 induces I-mediated resistance are indicated with a circle. Interactions where Avr1 suppresses I-2 or I-3 are indicated with an asterisk. N.I: not infected.
To confirm that the AVR1 gene is sufficient to trigger recognition by the I gene, we transformed AVR1 to a race 2 strain (Fol007) and a race 3 strain (Fol029) that do not contain AVR1 (Fig. 2B, lanes 4–9) and are virulent on I-containing tomato lines. Ten independent transformants (six of race 2 and four of race 3) containing AVR1 were unable to cause disease on I-containing plants (Fig. 3A, panels B and C, quantified in Fig. 3B), confirming the avirulence character of AVR1. In contrast to Avr3 , Avr1 is dispensable for full virulence towards plants that do not contain R genes against Fol (results not shown).
Avr1 suppresses I-2 and I-3-mediated disease resistance
Although all Fol strains possess an intact AVR3 gene, most race 1 strains nevertheless cause disease on plants carrying only the I-3 gene . One explanation for this is that Avr1 itself is involved in suppression of I-3 mediated disease resistance. To test this, we inoculated a plant line containing only the I-3 gene with the set of Fol strains described above. The results clearly show that Avr1 indeed has this suppressive activity: deletion of AVR1 in race 1 leads to loss of virulence towards I-3 plants (Fig. 3A, panel D, quantified in Fig. 3B), while introduction of AVR1 in race 2 or race 3 leads to gain of virulence towards I-3 plants (Fig. 3A, panels E and F, quantified in Fig. 3B). Furthermore, we discovered that Avr1 also suppresses I-2-mediated disease resistance (Fig. 3A, panels D and E, quantified in Fig. 3B). This means that the ability of some race 1 strains to cause disease on I-2 plants, as observed earlier , is likely to be caused by suppression of I-2 rather than loss of AVR2. In accordance with earlier observations using I-3 plants , we found that virulence due to suppression of I-2 and I-3 is partial compared to strains lacking the corresponding AVR gene (Fig. S1). It should be noted that not all race 1 strains are virulent on I-2 and/or I-3 plants ,, even though all contain AVR1 with identical sequences (results not shown). Apparently, suppression of R gene-based immunity by Avr1 is dependent on unknown factors in the genetic background of the fungus. Since suppression works in Fol007 (race 2) and Fol029 (race 3), the genetic background in which AVR1 is effective is not restricted to race 1 strains.
Possible function of Avr1
Our observation that Avr1 is not required for virulence to plants without I genes may be due to the existence of other effectors that are redundant for such an activity. Alternatively, the role of Avr1 is restricted to the suppression of I-2 and I-3-mediated disease resistance. A mechanistic explanation for the latter role could be that Avr1 interferes directly with Avr2 and Avr3. However, at least Avr3 accumulates in xylem sap and remains unaltered in the presence of Avr1 ,. A direct interaction between the two proteins could also not be demonstrated in vitro by pull down experiments (results not shown). Unlike bacteria, pathogenic fungi are not known to inject proteins directly into plant cells, but many are known to secrete small, frequently cysteine-rich, but otherwise unrelated proteins during colonization of plants . Avr1, like Avr3, falls within this group, the predicted mature protein having 184 residues including 6 cysteines and lacking homology to other proteins . The mode of action of most of these small secreted proteins has remained unclear. Molecular targets have been described for Avr2 and Avr4 from the leaf mold Cladosporium fulvum: Avr2 is a protease inhibitor  while Avr4 binds chitin in the fungal cell wall and protects it against attack by plant chitinases . These two proteins act in the apoplast to enhance fungal virulence, but others act inside plant cells . Uptake from the apoplast by plant cells has been shown directly for ToxA, a small secreted protein that acts as a host-selective toxin . This may also occur with Avr2, since I-2 is a cytoplasmic protein . Avr1, then, may interfere with the uptake of Avr2 and Avr3. Alternatively, it may be taken up itself and interfere with I-2 and I-3 or with signal transduction processes downstream of these R proteins (Fig. 4).
Arrows signify activation, lines ending in a cross bar signify suppression. Avr1 is synonymous to Six4, Avr3 is synonymous to Six1.
Implications for the evolution of Avr-R gene interactions
Suppression of effector-triggered (R gene-mediated) immunity has been observed in bacteria ,,. In plant pathogenic fungi, suppression of avirulence by unlinked loci has been demonstrated by genetics in rust fungi . In the flax rust fungus, two dominant alleles or tightly linked genes at the I (“inhibitor”) locus suppress – sometimes partially – either one (M1) or several (M1, L1,7,8,10) R genes out of 30 against flax rust ,. The flax rust inhibitor locus is not itself linked to avirulence. Here, we report the identification of a fungal avirulence factor that suppresses disease resistance conferred by two R genes.
Interpreting this phenomenon in terms of molecular arms races between plants and their pathogens , we envisage the following scenario. During evolution of the tomato-Fol pathosystem, I-2 and I-3 have evolved to recognize, respectively, Avr2 and Avr3. Since Avr3 is required for full virulence of Fol, evasion of I-3 recognition through loss of the AVR3 gene would entail a serious fitness penalty. This explains why all Fol strains analysed so far retained AVR3 ,. Point mutations in AVR3 preventing recognition have not been found either . A possible explanation for this is that the I-3 protein operates in accordance with the guard model, in which not the Avr3 protein itself but the effect it has on its virulence target is recognized . In any case, Fol has (partially) regained virulence towards I-3-containing plants by acquisition of AVR1, which, as shown here, suppresses the function of I-3. Subsequently, tomato responded to this ‘invention’ with the employment of the I gene, or the unlinked I-1 gene, to specifically recognize and respond to Avr1. Apparently, I and I-1 are themselves insensitive to the suppressive effect of Avr1 (Fig. 4).
The agricultural ‘arms race’ between Fol and tomato is different from the natural one because it is dictated by successive R gene deployment in commercial cultivars . The I gene from the wild tomato relative Solanum [Lycopersicon] pimpinellifolium was the first R gene to be introgressed into tomato cultivars to resist Fusarium wilt in the 1940s . At that time, Fol strains without Avr1 may already have been present in some locations, since I-breaking race 2 strains were quickly discovered  even though major outbreaks did not occur before 1960 . The I-2 gene, also from S. pimpinellifolium and directed against Avr2, was introduced in commercial cultivars in the 1960s to protect tomato against Fol race 2 ,. The combination of I and I-2 was effective for about two decades until the appearance of race 3 in both Australia and North America , which probably emerged from a race 2 background through selection for loss or mutation of AVR2. To combat race 3, the I-3 gene was introgressed from S. pennellii . From the results presented here, we deduce that the combination of I (or I-1) and I-3 may yield durable resistance of tomato to Fusarium wilt disease of tomato, since I-3 is directed against a virulence factor (Avr3) and I (and I-1) against the suppressor of I-3 (Avr1).
The molecular toolbox that is now gradually filling up (Avr1, Avr3, I-2) will help us to define host targets and evolutionary bottlenecks that govern the arms race in the Fol-tomato pathosystem. It also may allow development of new strategies for breeding plants with durable resistance against fungal pathogens.
Cracking Your Genetic Code
(Program not available for streaming.) What will it mean when most of us can afford to have the information in our DNA—all six billion chemical letters of it—read, stored and available for analysis? "Cracking Your Genetic Code" reveals that we stand on the verge of such a revolution. Meet a cancer patient who appears to have cheated death and a cystic fibrosis sufferer breathing easily because scientists have been able to pinpoint and neutralize the genetic abnormalities underlying their conditions. But what are the moral dilemmas raised by this new technology? Will it help or hurt us to know the diseases that may lie in our future? What if such information falls into the hands of insurance companies, employers or prospective mates? One thing is for certain: the new era of personalized, gene-based medicine is relevant to everyone, and soon you will be choosing whether to join the ranks of the DNA generation.
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CRACKING YOUR GENETIC CODE
PBS Airdate: March 28, 2012
NARRATOR: This is no ordinary flash drive. From a small company called Knome, it contains a complete digital record of a person's genetic code, all six billion letters of it.
NATHANIEL PEARSON (Knome, Inc.): Your D.N.A. is what makes you unique. It governed how you grew in the womb and how you look today. And, until now, only a few hundred people in the world have had a chance to see their whole genome and try to understand it.
NARRATOR: Few could afford the cost: $350,000, just three years ago, but that's changing.
FRANCIS COLLINS (National Institutes of Health): It's almost amazing to be able to say that each of us will have the chance to have our complete genome sequenced, for less than $1,000, in the next four or five years, but it's true.
NARRATOR: The result could be a revolution in medicine: using genetic information to diagnose and cure disease.
JOE BEERY (Noah and Alexis Beery's Father): If you go back and you look at some of the home movies that we took, and you see Alexis falling down, and you look at her now, you think, it's unimaginable that she was actually that same child. I really do believe that whole-genome sequencing really, really saved Alexis' life.
NARRATOR: But it could also lead to wholesale invasions of privacy and an ethical quagmire.
JAY ADELSON (23andMe Client): There's a lot of fear about, say, insurance companies or other professionals being able to access that data.
RUDOLPH TANZI (Massachusetts General Hospital): And then the company geneticist says, "He has an increased risk for cancer, okay? Just don't interview him he'll never know." Do you want that? Because that is potential reality.
NARRATOR: Thousands of years ago, the Ancient Greeks were given some famous advice: "Know thyself." Today, when those words are a biotech company motto, they present a new kind of challenge. Just how well do you want to know yourself in the age of personal genomics?
Up next on NOVA: Cracking Your Genetic Code.
A few years from now, you may boot up your tablet to find a life-changing report: a report on your own, personal genetic code, on the thousands of genes that spell out your body's instructions. Deciphered, your genes will reveal your risks for one disease after another, those you may get yourself and may pass on to your children.
How will it feel to have this information? You may find out sooner than you think.
GREGORY STOCK (Author, Biophysicist): We're entering an era of unprecedented self-knowledge. We are really beginning to come to understand the living processes that constitute ourselves, where we can begin to intervene to take control of our own future.
FRANCIS COLLINS: Genomics offers us the chance to look, in the most precise way, at what the causes of illness are and how to prevent and treat illnesses with that information. And we have that opportunity, now, in front of us.
NARRATOR: This could be your future:a new kind of personalized medicine based on your genetic code, one that predicts risks, so you can stop diseases before they appear, if there's a way of stopping them.
RUDI TANZI: But what if you can't? What if you have a gene mutation that says, doesn't matter how you live your life, doesn't matter what drugs you take, you will get this disease and probably before 50 years old.
CATHERINE ELTON (Journalist): Not everybody can handle genetic testing. And this information affects the way you live the rest of your life, if you are going to get a disease.
NARRATOR: But while some sound notes of caution, the science is rushing ahead and is now taking on medical challenges once thought impossible.
Consider Andrew Schmitz, a bubbly five-year-old, who has no idea his life hangs in the balance.
PAULA SCHMITZ (Mother of Andrew Schmitz): It started with high fevers and joint pains. And then, July he had his first stroke. And then he had two in October and one in November that required brain surgery. And then, his last one, number five, was a week ago.
NARRATOR: Andrew is at the center of a medical mystery. His parents have consulted dozens of specialists, but so far his symptoms defy diagnosis. He gets steroids to calm his immune system and has been on and off chemotherapy. Nothing seems to work.
At Children's Hospital, in Milwaukee, Andrew's pediatrician, Dr. Sheetal Vora, assesses his condition and the toll being taken by the drugs used to treat him.
SHEETAL VORA (Children's Hospital of Wisconsin): It pains you, because I have been there with this family from the beginning, and I have seen the ups and downs and told them the brutal truth, that you use all these medications, but they also can have their side effects.
NARRATOR: Desperate for a diagnosis, Dr. Vora has brought in geneticist Howard Jacob.
HOWARD JACOB (Medical College of Wisconsin): Right now, we don't know what is the cause of his disease. It's possible that it's environmental, or it's possible that he had some type of an infection. In general, though, somebody else should have it. Why doesn't anybody else have it in the family? What about in the community? So, a more plausible explanation is that it's genetic.
So if it happens to fall within a gene…
NARRATOR: If Jacob is right, there's a chance that Andrew's condition could finally be diagnosed, opening up the possibility of a cure.
HOWARD JACOB: So we will do everything we can to comb through his genome and see what we can find.
NARRATOR: To fulfill this promise, Jacob will be putting Andrew in a select group, those who have had their genomes, that is, all the genetic material contained within their cells, read out, letter by chemical letter, six billion, in all.
To start the process, a nurse draws Andrew's blood. The next day, it arrives at Illumina, one of a handful of companies that reads, or sequences, genomes.
In the lab, the blood is processed to extract its genetic material. As proteins and fats are washed away, delicate fibers clump together. This is D.N.A., life's master molecule. Next, the D.N.A. is sheared into fragments, making it easier to sequence.
It is such a complex task that sequencing the first human genome took 13 years, three billion dollars and hundreds of scientists. When the first draft was finished, in 2000, it was hailed as one of humanity's great achievements.
ERIC LANDER (Broad Institute of M.I.T. and Harvard): This is all the instructions there are, telling you all the tricks cells use to actually go from being a single cell to a whole grown up individual. All those recipes are written in exactly the same language.
NARRATOR: A language whose alphabet consists of four chemicals:each known by its initial:A, T, C and G. Strings of these chemical letters spell out some 20,000 genes, on 23 pairs of chromosomes. Genes code for proteins, molecules that do most of the work in our cells and help build parts of our body, from muscles to hair.
And, in the world of genes and proteins, spelling counts. If D.N.A. is copied incorrectly or damaged, spelling errors, known as variants or mutations, crop up.
NATHANIEL PEARSON: Now, when you change the spelling of a gene, sometimes it drastically changes the way that a protein functions, and those are the kinds of changes in the genome that we really look to when we are trying to trace disease, when we are trying figure out what spelling variant in the genome explains, for example, why this child is sick.
NARRATOR: And that's what Howard Jacob will search for. Convinced that a misspelled gene underlies Andrew's condition, he will comb through the boy's genome to find it. But even if he does, it's still a gamble.
HOWARD JACOB: The chances are pretty high that we're going to find something that there's nothing we can do about it. And that's where, I think, a lot of times, people worry about, well, if you can't change it, why do it? And we believe that providing an answer to the family does have merit to the family, even if we can't help his outcome. And so, here, we've decided that it's better to go look and potentially fail by looking than to not have looked and missed an opportunity to succeed.
NARRATOR: As advances in technology drive down the cost of D.N.A. sequencing, it's becoming accessible, not just to the sick, but the curious. And a handful of companies have arisen to meet the demand. They don't offer whole-genome sequencing yet, more of an economy-class genome scan.
One of the best known is the Silicon Valley start-up, 23andMe, co-founded by Anne Wojcicki.
ANNE WOJCICKI (23andMe): From day one, when we started this company, our goal has been, "How do we make genetics accessible?" And we started off with the genotyping technology, because it's a fabulously robust technology. It's incredibly reliable, and, most importantly, it's inexpensive.
NARRATOR: Human genomes are 99.9 percent identical, but by analyzing the D.N.A. in your spit, 23andMe will show you a million sites in your genes where the spelling sometimes differs between people. These one-letter variants may predispose you to certain traits and diseases.
A million letters may sound impressive, but that's far less than one percent of your D.N.A.
JONATHAN ROTHBERG (Inventor of Next Generation Sequencing): Genotyping is not D.N.A. sequencing. Genotyping is identical to looking at 100 words in a 600-page novel and believing you know everything about Tolstoy.
NARRATOR: Genotyping can explain odd traits, such as why some people find Brussels sprouts bitter, but when Jay Adelson wanted to know his chances of getting the brain disorder known as Parkinson's disease, he found the results far less clear.
JAY ADELSON (23AandMe Client): There is something called an odds calculator. And that odds calculator says I have roughly a 60 percent chance of contracting Parkinson's disease. And my father has it, but his parents and his grandparents…no one had Parkinson's disease. So, while it's a genetic trait that passes down, it doesn't necessarily mean that I'm going to contract it. And so, I spent a lot of time trying to understand what this meant.
TOM MURRAY (The Hastings Center): Almost all the evidence we're going to get will be, not hard, determining facts about our futures, but it'll be probabilistic information. It will tilt the odds one way or another.
ROBERT GREEN (Brigham and Women's Hospital): And we're not very well prepared, as a society, to, sort of, negotiate those risk elements. And some people will over-interpret those risks, and they will rush out and try to get diagnostic tests, or they'll try to get surveillance test to try to help them interpret what this information means or doesn't mean.
NARRATOR: Given such concerns, critics argue that genetic information should only come from a medical professional. Anne Wojcicki disagrees.
ANNE WOJCICKI: As of today, we're the only company that allows you to go direct, to the Web site and not require you to go through a physician. And, again, I think this is really core to our belief that this is your genetic information and something that's fundamentally about you and that you should have the right to get access to that information.
NARRATOR: Even the head of the National Institutes of Health, Francis Collins, decided to take the genomic plunge. He submitted his D.N.A. to three genotyping companies, including 23andMe.
FRANCIS COLLINS: I was a little on the cynical side about, "Yeah, yeah, these are early days, and how do I know they even got the results right?" But opening up that Web site, and beginning to look down the list of things where I turned out to be at higher than average risk got my attention.
NARRATOR: All three companies agreed Collins was at a substantially increased risk for getting type 2 diabetes.
FRANCIS COLLINS: And it got me motivated. I am 27 pounds lighter today than I was two years ago, and I'm working out three times a week. And the chance to have a little bit of a prediction about your future, as imperfect is it is right now—and it's very imperfect—could still be a teachable moment.
NARRATOR: But some of the results were contradictory.
FRANCIS COLLINS: Prostate cancer was the most glaring example. One company said higher than average risk one said about average the other said lower than average risk.
NARRATOR: So what's going on? According to neurogeneticist Rudy Tanzi, companies often look at different parts of genes and make predictions based on incomplete data.
RUDY TANZI: And even when we have that full set of genes where these variants increase your risk and these variants protect you, knowing how they work together or independently to come up with a real number? Good luck.
And remember, most of those variants are going to work together with your lifestyle. They're not guaranteeing anything. It depends on how you eat, do your exercise.
DAVID ALTSHULER (Broad Institute of M.I.T. and Harvard): I always wonder, is it possible that the person that is told they are not at high risk will do less and maybe be harmed?
RONALD GREEN (Dartmouth College): Oh, I don't have a cardiac problem, genetically. Now I'm going to go out and eat rich foods night and day, and so on. So, information is always hard to handle.
NARRATOR: One example is a piece of genetic information so potentially disturbing that even a founder of modern gene science refused to take a chance on running into it.
KEVIN DAVIES(Author, $1,000 Genome): James Watson, the man who co-discovered the double helix of D.N.A., one of the very first people in the world to have his genome completely sequenced, even before that was done, he said to the scientists who were doing the sequencing, "There's one gene I don't wish to know anything about."
NARRATOR: Known as ApoE4, on chromosome 19, it has been associated with late onset Alzheimer's, the leading cause of dementia in the elderly. The variant increases one's risk three- to tenfold.
RUDI TANZI: But what does that mean? What does it mean to say, "You have a tenfold-increase risk over someone who doesn't have it?" Can you then covert that into an absolute lifetime risk type of number to say, "Here's your percent chance of getting the disease?" No. Because you need to know what other genes work with ApoE. You need to understand how lifestyle is working together with ApoE.
NARRATOR: In fact, many people with ApoE4 never get Alzheimer's, while others with the disease don't have the variant.
ROBERT GREEN: I think the real key here is to disabuse people of the misunderstanding that genetics is wholly deterministic. In a few rare instances, there is a tight linkage between a particular mutation and a disease, but the vast majority of genetic information is largely probabilistic.
NARRATOR: Yet, some genes speak louder than others. Although we get two copies of most genes, one from each parent, certain dominant genes confer a trait of their own. And certain dominant disease genes, although rare, will eventually make you sick.
As a volunteer for the Huntington's Disease Society, Katie Moser works with people in this rare category, among them Meghan Sullivan.
Four years ago, Meghan was a high school student with everything to look forward to, but, with college, came a set of heartbreaking symptoms.
MEGHAN SULLIVAN (Huntington's Disease Patient): I was a perfectionist, but all my grades, my sophomore year, were Ds. I had no idea why, because, in high school, since 5th grade, I made the honor roll every single time.
NARRATOR: The reason for Meghan's plummeting grades, sudden movements and stumbling speech is a snippet of genetic code that repeats many times instead of a few, causing Huntington's disease. The mutation creates an elongated protein in the brain, which is as toxic to Meghan as it was to Katie's grandfather, who also had Huntington's.
And if he passed this gene to Katie's mother, Katie had a 50 percent chance of carrying it herself. For years, she wondered if she should get tested.
RUDY TANZI: In the case of a gene mutation that guarantees the disease, that's a real tough decision, because there, there's no hope. If you get the wrong answer, it's somewhat of a death sentence.
KATIE MOSER (Huntington's Disease Society): I decided to get tested for the gene, because I wanted to be able to plan my life financially, physically, emotionally. I wanted to know if someday I would start showing symptoms, but my family did not want to know.
NARRATOR: Tests confirmed that Katie will eventually get Huntington's disease, but knowing has had repercussions:dates who disappear relatives who won't speak to her, since they must now confront their own genetic status.
"The burden of knowing" is what journalist Catherine Elton calls it.
CATHERINE ELTON: In this age of personal genetic testing, it's not personal. People exist in families, and by the nature that you have tested, you are revealing this information to people who may not want to know.
NARRATOR: Elton, herself, was offered a test for mutations in BRCA1 gene, linked with high chance of getting breast or ovarian cancer. Her mother, her grandmother and her aunt had their lives cut short by these diseases. If Elton had the mutation, she could minimize her risks or more by having her breasts and ovaries removed. If she wanted children, she would have to have them before surgery.
CATHERINE ELTON: I didn't want those results. I didn't want to have that in the back of my mind and maybe make me settle for the wrong guy and rush into having kids before I was ready. And I think there is a real fine line between avoiding death and ruining your life.
NARRATOR: But Elton, too, has paid for her decision. In 2008, while pregnant with her second child, she was diagnosed with breast cancer. Yet, despite the ordeal of surgery and chemotherapy, and the risk the cancer might come back, she's convinced she did the right thing by not getting tested in her 20s.
CATHERINE ELTON: If I had made those decisions at 27, I can't even believe what I would have missed out on. And as the technology becomes more accessible, people just think, "Well, this is what you do." But I happen to believe that some of the costs of knowing our genetic destiny can outweigh some of the benefits.
NARRATOR: While Catherine Elton sees genetic information as potentially damaging, others see her disease-causing variant, BRCA1, as one of the first you can do something about, a so-called "actionable" gene.
LEROY HOOD (Institute for Systems Biology): These are genes where, if you know the patient has a variant in that gene, of a particular type, you can actually counsel them on things that will improve their wellness.
NARRATOR: One of these variants causes deadly blood clots, but needn't, if you avoid long periods of immobility or take blood thinners. Another variant tells us we could fall victim, even in our teens, to a heart attack.
NATHANIEL PEARSON: As a healthy adult, if you learned about one of those variants and found out you carried it, how would that change your life? Well, you might actually invest in defibrillator machines for your home or your workplace. You might actually change your vacation plans, in terms of whether you want to do really strenuous sports activities or shock yourself by jumping into cold water.
NARRATOR: So far, scientists have found about 200 actionable genes, including one that boosts your chance of colon cancer by age 45.
LEROY HOOD: So, what can you do about that? Well, if you start colonoscopies at 25 or so, you can actually keep people free of the disease.
RUDY TANZI: If you look at a personalized-medicine approach, the mantra is "early prediction, early detection." You want to know pre-symptomatically if you're in trouble, so that you can start treatment to nip the disease in the bud stage, prevent it before it strikes.
FRANCIS COLLINS: And if you do get sick, and your doctor has to make a decision about how to treat you, there are going to be signals in your instruction book to say, "Not that drug. Use this one instead."
NARRATOR: Of course personalized medicine only works if we know the gene variant responsible for a condition.
Back at Milwaukee's Children's Hospital, the search for the genetic cause of Andrew's illness is beginning. His decoded genome has arrived from Illumina. Sequencing took 45 days and cost $7,500. The next challenge, and the major expense, is figuring out what it all means.
To find Andrew's variants, Jacob's team will compare his genome with thousands of others, but mainly with the reference genome sequenced by the Human Genome Project.
DAVID DIMMOCK (Medical College of Wisconsin and Children's Hospital of Wisconsin): So this, here, is the reference sequence.
HOWARD JACOB: And so the computer's the first pass, it basically goes through and asks a question at each point across Andrew's D.N.A.:"Are you the same or different from the reference?" And when we see a difference, we then ask a question:"Is that difference meaningful?"
NARRATOR: Meaningful in that the variant must be unique to Andrew, and a possible cause of disease. But as the list of suspects shrinks from three million to a few thousand, success proves elusive.
Meanwhile Paula and Mike Schmitz keep Andrew's life as normal as possible.
MIKE SCHMITZ (Father of Andrew Schmitz): We had hoped that he would progress a little better with his rehab, with his walking. And it's hard to live every day with the anxiety of not knowing what's next, you know? And with Andrew, there's always something next. But, there is hope.
NARRATOR: One source of this hope is another Wisconsin boy. From age two, Nicholas Volker struggled with a condition that ate holes in his intestines. When treatment after treatment failed, Nick's doctors turned to Howard Jacob and gene testing.
HOWARD JACOB: That's for your birthday. Shall we open it?
NARRATOR: Now, nearly two years later, Nick is paying Jacob a visit.
HOWARD JACOB: Ask the question, "How many Ph.D.s does it take to assemble a Green Lantern?"
NARRATOR: Nick is the model for the success of this approach. His illness was traced to a gene on chromosome X, called XIAP, linked with immune disorders. A single letter was out of place:a G that had mutated into an A.
NICHOLAS VOLKER (Patient of Howard Jacob): I want to do the tank.
HOWARD JACOB: You want to do the tank?
HOWARD JACOB: I was afraid you were going to say that.
We found one single letter change in this gene, XIAP, which now was unique. Nobody else has the same variation that Nick has. And that's meaningful, because that means that letter is so important, that anybody who would have had this particular variation would die.
NARRATOR: This single variant caused Nick's symptoms, but a transplant, giving him a donor's immune system, without the misspelling, appears to have saved him.
In Boston, genomics is also being used in the battle against better known conditions, like cystic fibrosis, or C.F.
Michael McCarrick, age 29, knows its ravages first hand.
MICHAEL MCCARRICK (Cystic Fibrosis Patient): How I've experienced my decline has been sort of…in the beginning of my life, I played a lot of sports, and I had fun playing a lot of sports, and then, suddenly, I couldn't run, but I could walk long distances, and now it is like walking, itself, is difficult.
NARRATOR: Like many C.F. patients his age, Michael is facing end-stage lung disease.
AHMET ULUER (Children's Hospital Boston): Where Michael is right now and what he is dealing with is the torture we don't like to watch our patients go through, the struggle for that next breath.
NARRATOR: Michael may need a lung transplant, but Dr. Uluer hopes a new, gene-based drug will save him instead. Called Kalydeco, it targets a mutation found in four percent of C.F. patients.
MICHAEL MCCARRICK: Every drug can have a side effect. But I am hoping this one is free and clear, because who needs an extra problem? So, hopefully it's a magic bullet.
NARRATOR: The development of a drug for cystic fibrosis is especially gratifying to Francis Collins. Decades ago, Collins and a team of scientists, using rudimentary technology, set out to find the genetic defect behind the disease.
FRANCIS COLLINS: Back in the 1980s, this was like looking for a needle in the haystack, in the dark, with thick gloves on. And one day, in the spring of 1989, the data came across showing that individuals with cystic fibrosis were missing just three letters of that code.
NARRATOR: Just three letters, that is, from each copy of a gene called CFTR on chromosome 7. Because the mutation is recessive, only those who inherit a copy from both parents get the disease.
While we now know 1,800 different mutations in this gene can cause C.F., Collin's team found the main one, truly his needle in the haystack.
FRANCIS COLLINS: When this was announced, in August of 1989, the excitement was palpable. And I think many of us thought maybe this is the launch of a therapeutic effort that could happen pretty quickly.
NARRATOR: In reality, a clinical breakthrough would take another 20 years of research and hundreds of millions of dollars.
Some of the most successful work has been done at Vertex Pharmaceuticals, in San Diego, where scientists are trying to fix the defective protein made by the C.F. gene.
The normal protein creates an opening for salts to move across cell membranes, keeping them moist enough for tiny hairs called cilia to beat and remove mucus.
FREDRICK VAN GOOR (Vertex Pharmaceuticals Incorporated): In C.F., the cilia are not able to clear out the bacteria and the mucus, and this leads to chronic infection. So the first challenge was to find molecules that help the protein work better.
NARRATOR: Drawing on a vast chemical library, the research team employed a small army of robots to test 600,000 compounds on cells taken from C.F. patients. To date, two drugs have shown the most promise one of them:Kalydeco. When tested on C.F. cells, it helped the protein function, allowing salts and fluids to flow across membranes. Van Goor could watch the result:cilia beating, clearing away mucus, while untreated cells languished.
FREDRICK VAN GOOR: It was an exciting moment, where we really felt we are on the right track, designing drugs to fix a specific problem in a protein caused by a mutation.
NARRATOR: Whether Kalydeco will work for Michael McCarrick, whose lungs are severely damaged, remains unclear. But younger patients, like Paul Glynn, are seeing a world of difference.
PAUL GLYNN (Cystic Fibrosis Patient): It's a lot easier, ⟊use I am not coughing, and I can breathe easier. And then my weight, it's been going up.
NARRATOR: Since taking the drug, Paul has gained 12 pounds, enough to make the local football team. For him, the hope is that C.F. will become a manageable disease, and regular hospital visits a thing of the past, which may even make the drug cost-effective, despite its price tag:as much as $294,000 year.
In Boston, meanwhile, at Massachusetts General Hospital, doctors are using new gene-based drugs to target the most common disease of the genome: cancer.
Tom Garpestad is a 50-year-old building contractor. He was stunned to learn he had the skin cancer called melanoma.
TOM GARPESTAD (Melanoma Patient): Melanoma is the cancer that doesn't act like cancer. I had no symptoms, no weight loss, no night sweats. I felt perfectly fine, up until the point that my neck started bothering me.
NARRATOR: Scans revealed that the cancer had spread from his skin to his neck, lungs and liver.
KEITH FLAHERTY (Massachusetts General Hospital): Patients who have melanoma that has spread to other parts of the body from the primary skin site, have, from the time of initial diagnosis, typically a year or less. Tom had already had metastatic melanoma for sufficiently long that, as he walked in the door to our clinic, he was down to a month or two.
TOM GARPESTAD: I talked to my brother, who is a doctor, and he started telling me how severe melanoma is. You know, the average life expectancy of somebody with metastatic melanoma is nine months.
TODD GOLUB (Broad Institute of M.I.T. and Harvard): Research labs weren't even working on it that hard, because it seemed to be completely intractable nothing would ever work. And this all changed, almost overnight, with the sequencing of the melanoma genome.
ERIC LANDER: The idea of sequencing a cancer…it's as big as the human genome. Each cancer cell has an entire human genome in it, just mutated in various ways. And genomics told us all that there was a mutation in many melanomas, in a specific gene that goes by the funny name, BRAF. And it led to the idea that if you could inhibit this BRAF, you might be able to stop the melanomas.
NARRATOR: Luckily for Tom, gene sequencing revealed he had the BRAF mutation. He could now join a clinical trial of a new drug designed to neutralize its effects.
TOM GARPESTAD: The cancer was getting very aggressive. And then they started me on the BRAF medicine, and, within a week, I could feel the tumors shrinking.
NARRATOR: The BRAF mutation results in a defective protein that signals cells to divide uncontrollably. The drug binds to this protein, stopping both the signal and the cancer. Unlike chemotherapy drugs, this one kills only cancer cells, nothing else.
PET scans of patients reveal tumors that once riddled bodies, shrinking or vanishing within weeks.
KEITH FLAHERTY: We had a situation in a disease that was never responsive to therapy 90-plus percent of the time. To have that turn around and have it be 90-plus percent of the time that the treatment worked, that's when we knew we had completely crossed into uncharted territory.
NARRATOR: Only two months after lying, near death, in the hospital, Tom was back to his old life.
TOM GARPESTAD: People close to me, seeing me back on the job, just getting out there and doing things again? It was amazing. I was always thinking, "Hey, how sick was I two months ago? Look at me now."
NARRATOR: But that doesn't mean the war is over. To Keith Flaherty's dismay, scans show melanoma returning in many patients. Cancer cells, like viruses and bacteria, can evolve to resist drugs, even those that target genes. But now, the ability to compare cancer genomes before and after treatment is allowing scientists to see how resistance develops.
ERIC LANDER: The goal is to use the cancer genome itself to tell us how to defeat cancer, tell us what's wrong in a cancer and where we should hit it, tell us how it's becoming resistant and how we can block it.
NARRATOR: With resistance, new mutations arise in melanoma tumors, and once again defective proteins ignite the cancer.
So, now, Tom is taking a second drug, which targets a mutation linked to these relapses.
Eight months after starting his treatment, he awaits the results of a new set of scans.
DONALD LAWRENCE (Massachusetts General Hospital): The scans do show some re-growth, but only in a limited area. In all the other areas where we have seen signs before, things look great.
NARRATOR: Afterwards, Tom takes in the news.
TOM GARPESTAD: I probably wouldn't be alive right now, you know? So, I mean, I'll take, I'll take the eight months. You know, I mean, it's still a miracle drug, I think.
NARRATOR: At present, patients like Tom are getting between two and 18 extra months of quality life from the new treatment. But genomics is also helping those for whom there are no targeted drugs.
Thousands of breast cancer patients are taking a gene test, which tells them how aggressive their cancer is, and whether they need chemotherapy or can safely skip it.
The hope is by studying cancer genes, we'll come up with a cocktail of drugs like those used to treat tuberculosis or H.I.V., to cure cancers or keep them in check.
TODD GOLUB: The big difference, though, is that now, really for the first time, we can think about the right combinations of drugs, based on the genome, based on the science of what's going on inside the cancer cell.
ERIC LANDER: This is not a project for my life this is a project for my kids this is a project, ultimately, for their kids. But if, in the course of this century, we have a complete roadmap of what a cancer knows how to do, that will be a mind-boggling advance in medicine.
NARRATOR: Another extraordinary advance would be to eliminate inherited diseases before birth.
We're already taking the first steps:by fertilizing an egg and producing an eight-cell embryo, you can pluck off one of those cells and analyze its genes. Then you can screen that embryo for a host of diseases, using a technique called "preimplantation genetic diagnosis," or P.D.G.
MARK HUGHES (Genesis Genetics Institute): When we first started performing this technology, I think our biggest worry was, "Aren't we going to create some kind of a birth defect that is maybe even worse than the disease we're trying to avoid?" And what we learned was that the embryo doesn't seem to mind.
NARRATOR: At the Genesis Genetics Institute, Mark Hughes and his team are using P.G.D. to test embryos for mutations that can give rise to over 300 diseases, including Huntington's and cystic fibrosis.
Only embryos free of certain mutations are implanted in the mother.
MARK HUGHES: So this is the mutant gene, and we have the normal gene over here.
Tens of thousands of healthy babies have been born to couples who otherwise would have been afraid to have a child, because the disease that they were at risk of giving their child was so severe.
NARRATOR: P.G.D. can also be used to test for traits like gender, leading ethicists to ask if designer babies are in our future.
RONALD GREEN (Dartmouth College): I think we are going to see people using genetics to select traits. Some of it will be relatively benign:"I want a child with this hair color or eye color."
GREGORY STOCK: As to where our ability to really intervene in our own living processes is really going to lead us, we simply do not know. And that is the promise and that is the threat of this period.
NARRATOR: Some fear P.G.D. could even lead to a new kind of eugenics and the sort of genetic elite depicted in the movie GATTACA.
DOCTOR (GATTACA Film Clip): Your extracted eggs, Marie, have been fertilized with Antonio's sperm. After screening we are left with two healthy boys and two very healthy girls. I have taken the liberty to eradicate any prejudicial conditions:premature baldness, myopia, alcoholism and addictive susceptibility, propensity for violence, obesity, etc.
NARRATOR: Fortunately, the traits a genetic elite would want—intelligence, physical ability, even height—are so complex, scientists assure us we won't be selecting them in embryos anytime soon.
ERIC LANDER: Trying to predict height, well, we already know there's at least 180 genes involved in height, and each contributes a little bit. It isn't going to be easy to go take some embryo and sort out which of these 180 are in which of these forms. You really, really, really want to have a tall person, go marry a tall spouse. It's just more efficient.
NARRATOR: Even so, GATTACA raises real concerns.
RONALD GREEN: One of the negative implications of this new genetic knowledge is that we're going to start thinking of ourselves more in genetic terms than we ever have before. Do I want to date that individual? What's her genetics? There's always been a tendency to engage in deterministic genetics, and I think scientists and medical people and educators must make very clear the limits of that point of view.
PATRICIA WILLIAMS (Columbia University): We are deeply affected by the kind of food we eat, the air we breathe, by the kind of good luck or bad luck that shapes our lives, like education and money, and by the real romance of simply falling in love with an unlikely partner. We narrow our vision if we focus or fetishize upon genetics.
NARRATOR: But as the cost of a sequenced genome falls, new generations may not get far into the world without one.
JONATHAN ROTHBERG: And I envision a day when every child is born, they prick that child's heel, and that D.N.A. from that child is decoded right at birth.
NARRATOR: For 14-year-old twins Noah and Alexis Beery, sequencing at birth could have made a world of difference. Today, it's hard to imagine that just two years ago, Alexis was fighting for her life.
ALEXIS BEERY: The only really vivid memory that I have of that is just…simple as just seeing red, blue and white lights just flashing everywhere. That's definitely one of my, like, number one, kind of like, nightmares that I still have.
JOE BEERY: After Alexis and Noah were born, they didn't reach any of their milestones. They didn't crawl on time, they didn't walk on time. And we knew, pretty early, that something wasn't right.
NARRATOR: The twins were diagnosed with cerebral palsy. Then, at age five, Alexis got worse.
RETTA BEERY (Noah and Alexis Beery's Mother): She started losing more and more ability to walk during the day. She started losing ability to sit up by 10:30, 11:00 in the morning. She could no longer swallow by that time. And so, that's not indicative of cerebral palsy.
NARRATOR: Retta began doing research. One day, she came across a rare condition that mimics cerebral palsy, a condition that could be treated with the brain chemical dopamine. Dopamine is crucial to our bodies' ability to move.
And the morning after Alexis took it, she woke up to a new world.
JOE BEERY: She was walking, she was talking, she was using her arms, she was whistling.
NARRATOR: Noah also responded. Yet the twins still had health issues, especially Alexis.
RETTA BEERY: We actually almost lost her on a couple of occasions. We had paramedics coming into our house, trying to get her breathing, and we were back into that world of unknowns.
NARRATOR: Until, that is, Joe asked his new employer, a biotech company, for help getting the twins' genomes sequenced.
JOE BEERY: We discovered, through sequencing, that there was a second problem associated with a rare mutation that they had.
NARRATOR: The mutation was suppressing yet another brain chemical. Another drug solved that problem, too.
JOE BEERY: Whole-genome sequencing really saved Noah and Alexis's life. Had that happened at birth, we would have 15 years less pain and suffering and probably millions of dollars of cost.
NARRATOR: Yet sequencing at birth may have its downside.
RONALD GREEN: One of my concerns is the testing of children raises many questions, including stigmatization. "Oh, I'm not going to let Mary play soccer, because she's got this cardiac risk." So we do have a problem of invasion of the child's privacy.
NARRATOR: And what about your privacy? What if a company asks to use your D.N.A. for research, promising you'll remain anonymous.
PATRICIA WILLIAMS: Now the question is whether or not that anonymized information is truly anonymized, just because they take your name and your social security number off. At some point in the future, it may be that you don't need a social security number, that you don't need to give your name, because your genetic information will reveal you so precisely that we'll have to develop a whole new definition of what we mean by anonymity.
NARRATOR: And what if your D.N.A. reveals you're at increased risk for an incurable disease, say, Alzheimer's?
TOM MURRAY: Can a company that sells long-term care insurance ask you about that? And can they use that result to either affect your premiums or even deny you insurance, entirely because you're such a bad bet?
NARRATOR: In most states, the law already allows long-term care, disability and life insurers to discriminate based on genetics.
RUDI TANZI: I would not make one single base of my D.N.A. sequence available publicly, in a million years. There's too much risk. You don't know what's going to happen in the future with insurance. And think about your company going down the tubes, and now you have to get a new job. And, yeah, employers can't discriminate, but they happen to find out on your Facebook page, under D.N.A., click:there's the sequence.
NARRATOR: Everywhere you go, you leave a trail of genetic debris:when you cut your hair, enjoy a meal at a restaurant.
A crime that once seemed like science fiction has become possible with cheap, fast D.N.A. sequencing.
RONALD GREEN: It's been called "genomic hacking." That will be frightful. It will be used to impugn people. Somebody saying he should not or she should not be a candidate for the presidency, because she has the gene for depression. If somebody wants access to your genome for personal, romantic purposes, for economic reasons, for political reasons…in the future, if they want it, they'll go after it.
NARRATOR: Yet when illness strikes, privacy may seem a distant concern, and genomics your best hope.
In Milwaukee, after months of searching through Andrew's genome, finally, there's a discovery.
HOWARD JACOB: This is a list of genes that we pulled out. So yesterday, at noon, I got an e-mail from Liz saying, "I found a very interesting gene."
ELIZABETH WORTHEY: He's got two protein-coding….
NARRATOR: Geneticist Elizabeth Worthey has found something unusual in Andrew's genome.
HOWARD JACOB: There's two rare variants, one that's never been seen before, and one that's only been seen once, as far as we know.
The next question, then, is, "Does this gene look like it could explain part of Andrew's clinical features?" And in this case, the answer's yes.
ELIZABETH WORTHEY: Mutations in this gene have been linked with susceptibility to recurrent viral infections, which he had.
HOWARD JACOB: I will tell you that this is where we get both excited as scientists and nervous, then, as scientists, because you think you've found it. There's a big difference between thinking you've found it, to, "We've proved it."
NARRATOR: In fact, the suspect gene is soon ruled out, and the family learns the search must continue.
HOWARD JACOB: So, don't worry, until we tell you that we stopped looking, we haven't stopped looking.
PAULA SCHMITZ: Well, we really appreciate it.
NARRATOR: In a field this new, success and failure continually intermix.
In Michael McCarrick's case, the drug targeting his cystic fibrosis mutation helped, but his lungs were already so damaged, he died waiting for a transplant.
Yet Paul Glynn, who used to spend part of every fall in the hospital, is healthier than ever.
Tom Garpestad is in a kind of limbo, with some tumors shrinking, others growing.
As for the Beery twins, their only regret is not getting sequenced sooner.
FRANCIS COLLINS: We have focused so much of our energies on treating people with disease, often advanced disease, and much less effort on trying to prevent that disease in the first place. If we are going shift towards prevention, your genome sequence may be one of the most critical tools you could imagine.
NARRATOR: How can we balance the risks and benefits of this critical tool? This will soon be a question for all of us, as we take up the ancient challenge, "Know thyself," in the genome age.
CRACKING YOUR GENETIC CODE
For The Hastings Center
Research and development funded in part by The Greenwall Foundation.
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The Hastings Center
The Hastings Center is a non-partisan research institution dedicated to bioethics and the public interest. Here you'll find information on the latest research, along with forums, blogs, and advice on how to deal with difficult bioethical issues.
Genetic Testing and Screening
Do you have questions about genetic testing? This site gives a comprehensive background on many common forms of genetic analysis offered by the medical community, and probes ethical dilemmas from prenatal screening to direct-to-consumer genetic testing.
Personalized Medicine and Genomics
This website offers information on the ethical issues surrounding the use of genomics to tailor health care to the individual. It explores the complexities of genetically-based health care, risk assessment, and more.
Here you will find information on stored biological specimens (such as blood, saliva, and surgical tissue) and how they can be used in genetic and biomedical research. The site addresses the issues surrounding ownership of biospecimens, disclosing research results, and other ethical debates.
The Cystic Fibrosis Foundation
The Cystic Fibrosis Foundation is a non-profit organization that funds research towards finding a cure for cystic fibrosis.
The $1,000 Genome: The Revolution in DNA Sequencing and the New Era of Personalized Medicine
By Kevin Davies. Free Press, 2010.
Phages and their potential to modulate the microbiome and immunity
Bacteriophages (hence termed phages) are viruses that target bacteria and have long been considered as potential future treatments against antibiotic-resistant bacterial infection. However, the molecular nature of phage interactions with bacteria and the human host has remained elusive for decades, limiting their therapeutic application. While many phages and their functional repertoires remain unknown, the advent of next-generation sequencing has increasingly enabled researchers to decode new lytic and lysogenic mechanisms by which they attack and destroy bacteria. Furthermore, the last decade has witnessed a renewed interest in the utilization of phages as therapeutic vectors and as a means of targeting pathogenic or commensal bacteria or inducing immunomodulation. Importantly, the narrow host range, immense antibacterial repertoire, and ease of manipulating phages may potentially allow for their use as targeted modulators of pathogenic, commensal and pathobiont members of the microbiome, thereby impacting mammalian physiology and immunity along mucosal surfaces in health and in microbiome-associated diseases. In this review, we aim to highlight recent advances in phage biology and how a mechanistic understanding of phage–bacteria–host interactions may facilitate the development of novel phage-based therapeutics. We provide an overview of the challenges of the therapeutic use of phages and how these could be addressed for future use of phages as specific modulators of the human microbiome in a variety of infectious and noncommunicable human diseases.
Circadian rhythms in the cardiovascular system
Cardiovascular complications have higher incidence in the morning. Many different studies have connected the clock with cardiovascular function, including daily variation in blood pressure, and even response to aspirin [82, 145, 146]. Some studies suggest that pharmacological targeting of REV-ERB decreases atherosclerotic plaque burden in mice . On the other hand, other studies suggest that deletion of Bmal1 in myeloid cells increased monocyte recruitment and atherosclerosis lesion size . A recent study has shed light on a mechanism that may contribute to this phenomenon. The adherence of myeloid cells to microvascular beds peaks during the early active phase, which appears to be a consequence of peak cell recruitment to atherosclerotic lesions 12 h earlier . Winter et al.  showed that both the upregulation of cell adhesion molecules during the active phase by endothelial cells and the presence of immobilized chemokines (emitted by either endothelial cells or myeloid cells) on arterial vessels attract leukocytes into atherosclerotic lesions. Thus, the chemokine CCL2 (C-C motif chemokine ligand 2) and its receptor CCR2 (C-C motif chemokine receptor 2) are at the core of this daily pattern of leukocyte migration and adhesion to the lesions. Importantly, the authors found that timed pharmacological CCR2 neutralization caused inhibition of atherosclerosis without disturbing microvascular recruitment, providing a proof-of-principle treatment schedule for chrono-pharmacological intervention in atherosclerosis (Fig. 3).
Loss of Bmal1 results in an acceleration of aging and a shortened life span in mice . The cardiovascular system is among the systems affected by aging, with Bmal1 −/− mice being predisposed to developing atherosclerosis. Using an inducible knockout (iKO), Yang et al.  tested whether these age-related phenotypes remained if mice lost BMAL1 as adults. They found that both Bmal1 −/− and iKO models exhibit markers consistent with accelerated aging (ocular abnormalities and brain astrogliosis), behavioral disruption, and transcriptional dysregulation. This is consistent with the fact that conditional ablation of the pancreatic clock still causes diabetes mellitus . However, some other biomarkers of aging, including premature death in Bmal1 −/− mice, were not replicated in the iKOs . Among those, the predisposition for atherosclerosis appears to be reversed in iKOs . These data suggest that some of the cardiovascular phenotypes associated with Bmal1 depletion may result from Bmal1 function during development. Although it is clear that there is a link between the circadian clock and atherosclerosis, further dissection of the importance of BMAL1 and other clock proteins in this disease is warranted.
H3K27me3 destabilizes accessory chromosomes
We investigated the effects of loss of two important heterochromatin-associated histone modifications, H3K9me3 and H3K27me3, on chromatin organization, transcription and genome stability and characterized phenotypes of the deletion mutants. Loss of H3K9me3 allows relocalization of H3K27me3 in kmt1 deletion mutants, which has great impact on genome and chromosome stability, resulting in numerous large-scale rearrangements. In contrast, the genomes of evolved Δkmt6 and Zt09 strains revealed only few and relatively minor changes. Unexpectedly, the presence of H3K27me3 impacts chromosome stability by either destabilizing whole chromosomes in normal cells, supported by the high loss-rate in the reference strain compared to the Δkmt6 mutants, or by mislocalization as shown by the increased sequence instability in the Δkmt1 mutants. Taken together, enrichment with H3K27me3 in wild type cells is a main driver of mitotic chromosome instability.
We propose different scenarios for how chromosomes may get lost during mitosis and how H3K27me3 may be linked to these processes. For example, accessory chromosomes may not be accurately replicated whereby only one sister chromatid is transmitted. Alternatively, non-disjunction of sister chromatids during mitosis produces one cell with two copies and one cell lacking the respective chromosome. Previous cytology on Z. tritici strains expressing GFP-tagged CENPA/CenH3 protein suggested that core and accessory chromosomes may be physically separated in the nucleus . Previous studies showed that H3K27me3-enriched chromatin localizes near the nuclear periphery, and loss of H3K27me3 enables movement of this chromatin to the nucleus core in mammals and fungi [52,53]. Proximity to the nuclear membrane and heterochromatic structure can furthermore result in differential, and often late, replication timing [54,55]. Loss of H3K27me3 and the correlated movement to the inner nuclear matrix may alter replication dynamics of accessory chromosomes resulting in higher rates of faithfully replicated chromosomes and lower rates of mitotic loss (Fig 7).
In wildtype cells (left panel), H3K27me3 is localized in subtelomeric regions and on accessory chromosomes, directing those regions to the nuclear periphery and resulting in increased instability of these regions. Loss of H3K27me3 (middle panel) results in a relocation of former H3K27me3-enriched sequences to the inner nucleus and an increase of genome stability. Loss of the histone modification H3K9me3 enables H3K27me3 to spread, leading to mislocalization of H3K27me3, altered physical localization and chromatin interactions in the nucleus that fuel genome instability of these regions (right panel).
Heterochromatic regions, especially associated with H3K27me3, tend to cluster together and form distinct foci in the nucleus of Drosophila melanogaster visualized by cytology [56,57], and loss of H3K27me3 reduces interaction between these regions . We hypothesize that enrichment of H3K27me3 on the entire accessory chromosomes maintains physical interactions that persist throughout mitosis. This may decrease the efficiency of separation of sister chromatids resulting in loss of the chromosome in one cell and a duplication in the other cell. So far, we have focused our screening on chromosome losses but determining the exact rates of accessory chromosome duplications is necessary to test this hypothesis. Genome sequencing of Z. tritici chromosome loss strains revealed that duplications of accessory chromosomes can occur . Similarly, B chromosomes in rye are preferentially inherited during meiosis by non-disjunction of sister chromatids during the first pollen mitosis , indicating that deviation from normal chromosome segregation occurs. Accessory chromosomes are commonly found in natural isolates of Z. tritici, despite the high loss rates we demonstrated during mitotic growth . This observation implies the presence of other mechanisms that counteract the frequent losses of accessory chromosomes. Recent analyses of meiotic transmission showed that unpaired accessory chromosomes are transmitted at higher rates in a uniparental way [60,61]. We propose that H3K27me3 is involved in accessory chromosome instability and transmission both during mitosis and meiosis by influencing nuclear localization of chromosomes and thereby altering replication or transmission (Fig 7). Future analyses with fluorescently tagged core and accessory chromosomes and by chromosome conformation capture (Hi-C) will shed light on nuclear interactions and chromosome transmission dynamics. As not all accessory chromosomes, despite being enriched with H3K27me3, are lost at the same rate, we note that additional mechanisms likely contribute to accessory chromosome dynamics.
H3K9me3 loss allows invasion by H3K27me3 and results in genome instability
While loss of H3K27me3 resulted in only minor differences to wild type growth and, unexpectedly, rather promoted than decreased genome stability, we detected a high number of smaller (up to 30 kb) deletions and duplications, chromosome breakages and several gross chromosomal rearrangements linked to large duplications in the Δkmt1 mutants. Absence of H3K9me2/3 has been associated with chromosome and genome instability in other organisms [13,14,62,63]. Smaller deletions, duplications and chromosome breakages resulting in shortened chromosomes due to loss of chromosome ends that we identified in the Δkmt1 mutants, correlate with TEs, enriched with H3K9me3 in wild type. Replication of heterochromatin-associated DNA is challenging for the cell as repetitive sequences may form secondary structures that can stall the replication machinery . Consequently, instability of repeated sequences has been linked to errors during DNA replication [65–67]. Furthermore, the structural variation that arises depends on the mode of DNA repair following the DNA damage [68,69]. The structural rearrangements detected in the Δkmt1 mutants indicate that repair of double-strand breaks involves both non-homologous end joining and de novo telomere formation. We propose that the main factor for genome instability is replication-associated instability of repeated sequences subsequently promoting the formation of large-scale rearrangements (S11 Fig).
Not all breakpoints of rearrangements, especially of the large duplicated sequences, were associated with TEs, however. We found that duplicated sequences in the experimentally evolved Δkmt1 mutants fully or partially overlap with the duplicated regions of the Δkmt1 progenitor strain. This suggests that structural variations are subject to continuous rearrangements, resulting in rearrangements no longer directly linked to the initial event. We note that the rearrangements and genotypes we detected are the result of selection during our long-term growth experiments and thus do not necessarily reflect the full spectrum of rearrangements occurring in Δkmt1 mutants many additional structural variants may have disappeared quickly from the population or included lethal events.
Concomitant with loss of H3K9me3 in the Δkmt1 strains, we found relocalization of H3K27me3 to former H3K9me3 regions. A similar redistribution of H3K27me3 in absence of heterochromatin factors has been reported in plants and animals [70–72] and other fungi [22,27,73]. In N. crassa, redistribution of H3K27me3 in a Δkmt1 (dim-5) mutant background results in severe growth defects and increased sensitivity to genotoxic stress that can be rescued by elimination of H3K27me3, indicating that aberrant H3K27me3 distribution severely impacts cell viability . Although we did not see rescue of phenotypic defects observed in planta or in in vitro stress assays in the Δk1/k6 double mutants, the chromosome-loss rate was reduced compared to Δkmt1 mutants, suggesting a stabilizing effect when H3K27me3 is absent. We found that some breakpoints of the rearrangements in the Δkmt1 mutants without H3K9me3 also show enrichment with the invading H3K27me3. This finding also suggests that sequences associated with H3K27me3 are more susceptible to genome instability. Regions enriched with H3K27me3 have been shown to exhibit a high degree of genetic variability in form of mutations, increased recombination, or structural variation compared to the rest of the genome [21,23,30,31,74–76]. Experimental evolution in Fusarium fujikuroi showed that increased H3K27me3 levels in subtelomeric regions coincided with increased instability  and we previously detected a highly increased rate of chromosomal breakage under stress conditions in subtelomeric H3K27me3 regions in Z. tritici . These observations together with our findings strongly indicate that H3K27me3 plays a pivotal role in decreasing genome stability.
In summary, the presence of Kmt1 and H3K9me3 respectively, is essential to maintain genome integrity in this fungus. TE-mediated rearrangements may be involved in the genetic variability detected in Z. tritici isolates [78–80] and have been suggested as drivers of genome evolution in various species [81–83]. Our findings concerning the role of H3K9me3 for genome stability provide a basis for future studies focusing on the influence of heterochromatin on structural genome rearrangements using Z. tritici as a model organism. We found that, unlike for H3K9me3, presence and not absence of H3K27me3 is linked to genome instability. Surprisingly, loss of H3K27me3 does not result in dramatic changes of overt phenotypes and is also not clearly linked to transcriptional activation in Z. tritici. This allowed us to uncouple the transcriptional and regulatory effects of H3K27me3 from the influence on chromatin stability and will in the future result in further mechanistic insights on the influence of histone modifications on chromosome stability.
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Keywords: brucellosis, gut immunity, Brucella vaccines, intracellular infection, respiratory tract immunity
Citation: López-Santiago R, Sánchez-Argพz AB, De Alba-Nú༞z LG, Baltierra-Uribe SL and Moreno-Lafont MC (2019) Immune Response to Mucosal Brucella Infection. Front. Immunol. 10:1759. doi: 10.3389/fimmu.2019.01759
Received: 01 May 2019 Accepted: 11 July 2019
Published: 20 August 2019.
Luis F. Garcia, University of Antioquia, Colombia
Maryam Dadar, Razi Vaccine and Serum Research Institute, Iran
Sunil Joshi, University of Miami, United States
Copyright © 2019 López-Santiago, Sánchez-Argพz, De Alba-Nú༞z, Baltierra-Uribe and Moreno-Lafont. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.