Cytogenomics Disorders 2024 PDF

① CYTOGENOMICS DISORDERS DR. A. MOHSENI MEYBODI PATHOLOGY AND LABORATORY MEDICINE, LHSC, WESTERN UNIVERSITY Central Dogma OBJECTIVES:...

① CYTOGENOMICS DISORDERS DR. A. MOHSENI MEYBODI PATHOLOGY AND LABORATORY MEDICINE, LHSC, WESTERN UNIVERSITY Central Dogma OBJECTIVES: ↑ w 1. Understand key attributes of the human genome and the flow of genetic information 2. Define different types of genetic mutations (chromosome to nucleotide level) a. Define genomic disorders b. Understand how genomic diseases result from abnormal chromosomal number or structure, illustrated by examples 3. Describe the utility of cytogenetic techniques and indications for testing 4. Congenital abnormalities a. Differentiate between the terms congenital and genetic - - b. Define types of congenital abnormalities, illustrated by disease examples. c. Define a teratogen and examples of consequences in the developing fetus - - Recommended readings: "Robbins Basic Pathology" 11th Edition, 2023. Chapter 4 - read relevant sections; basic genetics textbooks (e.g. Lewin’s Gene X). THE HUMAN GENOME - ~3 billion base pairs introns get spliced that out ↑ L o Only 1.5% is protein coding; 26% intronic (within a gene, but not protein coding) o Rest of the genome contains various forms of repetitive sequences ▪ Some are critical for regulation of gene expression or spatial distribution - 23 chromosomes (22 autosomes and sex chromosomes (X and Y) o Gametes are haploid (n) o Somatic cells are diploid (2n), two pairs of homologous chromosomes (one from mom - maternal and one paternal) - from dad - Chromosome structure o Each chromosome is a single, continuous DNA double-helix o DNA is condensed and packed with histones (a special protein) as chromatin: - Mitochondria have a separate genome, outside of the cell nucleus. o 16kb in length with 37 genes o Maternally-inherited ↳ M in Mitochondria stands for maternally inherited- FLOW OF GENETIC INFORMATION - DNA to RNA to protein 3 Central Dogma - Cell division o Mitosis: somatic cells division, creates identical daughter cells 3 identical division o Meiosis: occurs only in the germline, where by diploid cells give rise to haploid 2n gametes 3 produces 4 different gametes - N o Both processes require proper pairing and chromosome segregation ▪ Errors can result in aneuploidies and other chromosome abnormalities - - Germline vs somatic genetics ↳ improper pairing ↳) inherited ↳ Standard ex. D⑭ ex. O D PATHOLOGY 3500/PATHOLOGY 9535 1 CYTOGENOMICS DISORDERS  Patar Syndrome Edwards syndrome GENETIC MUTATIONS AND GENOMIC DISORDERS - Aneuploidy: numerical abnormality in chromosome content ↓ down sundrome o Most aneuploidies are not compatible with life; trisomy 13, 18, 21 and various sex chromosome aneuploidies are the exception. Still, most of these conceptions will result in spontaneous abortion. o Examples: ▪ Autosomal aneuploidy: Trisomy 21 (Down Syndrome) ▪ Sex chromosome aneuploidy: 45,X (Turner Syndrome) - Structural abnormalities: abnormally formed chromosome * o Translocation: part of one chromosome is transferred to another. May be balanced/reciprocal (chromosomes swap material) or unbalanced (results in gain ABL gene-Ch9 encodes for tyrosine kinase involved in and/or loss of genomic material) : cell growth survival o chromosomes 9822 reciprocal between ▪ Examples: ~ is creates shortened version of chromosome Bergene-chan functions include signalling o gene W 22 : BCR-ABL/ t(9;22)q(q34;q11) in chronic myelogenous leukemia regulation betweenCh 18. t constitutional translocation (CML); -is they're locatedis An abnormal tyrosine kinase w/ constitutive activity where uncontrolled - (Always onl division cell inhibition Emanuel syndrome associated with the recurrent of apoptosis genome instability - , , t(11;22)(q21;q11) translocation. warn o Robertsonian translocation: translocation between the long arms of two acrocentric chromosomes; carriers have 45 chromosomes but are phenotypically normal, except for infertility o Isochromocome: translocation caused by horizontal split of homologous chromosomes, chromosome contains two copies of the short or long arm, and no copies of the other ! o Copy number variant (CNV) ▪ Deletion: loss of a portion of a chromosome insertions ▪ Gain: gain of a portion of a chromosome, occurs most commonly in · deletions tandem, but the additional material may also be inserted elsewhere in the · Duplication · genome inversion · ▪ Amplification: multiple copies of a portion of chromosome, most commonly in neoplasia. ▪ Examples:- 22r chromosome C O 22q11.2 deletion syndrome; position - x Smith Magenis vs Potocki Lupski syndromes o Inversion: segment of a chromosome is in an inverted orientation. May disrupt a gene or regulatory region. o Ring chromosome: may form after deletions of both ends of a chromosome ends fuse together > ▪ Example: Variant Turner syndrome - Sequence variants at the DNA level/resolution (More details next lecture) o Changes in nucleotide sequence ▪ Single nucleotide variants (SNV): one nucleotide substituted for another ▪ Insertion-deletion variant (indel): remove and/or insertion of two or more nucleotides ▪ Depending on nucleotide changes, sequence variants may or may not impact protein coding sequence CYTOGENETIC TECHNIQUES - Karyotype: examine number and structure of chromosomes within a cell PATHOLOGY 3500/PATHOLOGY 9535 2 CYTOGENOMICS DISORDERS  o Test indications: aneuploidy detection, infertility and recurrent pregnancy loss - FISH: enumerate a particular locus, detect specific rearrangements ① ② o Test indications: prenatal aneuploidy detection, targeted oncology testing - Microarray: genome-wide analysis of copy number variants ② o Two types:①Array comparative genomic hybridization (aCGH) and Single Nucleotide Polymorphism (SNP) microarrays o Test indications: multiple congenital anomalies, intellectual disability Fibrosis ex Trisomy 21 ex Cystic CONGENITAL ABNORMALITIES:.. - [ - L - Congenital (present at birth) vs genetic (determined by genes) o 3% of newborns have a congenital abnormality; ~55% caused by genetic disorders - Types of congenital abnormalities o Malformations: result from intrinsically abnormal development process. Can be isolated or there may be coexisting malformations of multiple tissues/organs. Usually multifactorial. ▪ Examples: Polydactyly and syndactyly, spina bifida, congenital heart ) extra fingers/toes Spinalcordspine Is structural abnormalities of the is her disease. ↳ Is toes/fingers fused together o Deformations: result from extrinsic disturbance of development by mechanical forces ▪ Examples: small uterus, large fetus, oligohydramnios, positional fluid during abnormalities of the feet. preg ↳ Amniotic , is abnormally low o Disruptions: result from extrinsic disturbance in morphogenesis, disturbance of - a previously normal development process. Not genetic. ↳ cells specific tissues & organisms develop shapes , structures their , a ▪ Example: Limb amputation due to an amniotic band. - - amniotic tears creating fibrous bands > when sac , wrap around that fetrs Sequences: multiple congenital anomalies that results from secondary effects of a can a - single localized aberration in organogenesis (could be malformation, deformation or - disruption) S organs form from embryonic cells in developing organism a - Syndrome: a characteristic association of several anomalies. problems ~ - Teratogens: agents that produce congenital - malformations. Can be from various sources, including: ↳ born with o Congenital infections (e.g. maternal rubella) o Drugs and chemicals (e.g. thalidomide) o Maternal factors (e.g. diabetes) o Ionizing radiation PATHOLOGY 3500/PATHOLOGY 9535 3 CYTOGENOMICS DISORDERS ② IMMUNITY AND IMMUNE DISORDERS DR. CHRISTOPHER HOWLETT PATHOLOGY AND LABORATORY MEDICINE, LHSC, UNIVERSITY CAMPUS OBJECTIVES: Part I: Biology of the Immune System After completing this review, you will be ofable to: ~ Your immune system the study your immune system o Define immunity and immunology. - o List the general differences between the innate (natural) and acquired (adaptive) immunity. o List the cellular, biological, and chemical components of the immune system, innate and adaptive. o Describe the morphological characteristics of the cellular component. o Identify the different functions of each cell type and understand the collaboration of different cells, systems and tissues to provide immunity to the body. o Describe the mechanism of action of the complement system in the immune response. o Describe the structures and functions of lymphocytes, including all subtypes. Part II: Hypersensitivity Reactions After completing this section, you will be able to: o List the four major types of hypersensitivity reactions. o List the cellular components and antibody(ies) involved in each reaction. o Understand the mechanism of action in each reaction. o Differentiate between cell-mediated and humoral reactions. Part III: Immunodeficiency and Autoimmune Diseases After completing this section, you will be able to: o List causes of primary and secondary immunodeficiency. o List common manifestations encountered in patients with immunodeficiency diseases. o Define autoimmune disorders and explain how autoimmune disease may develop (aetiology). RECOMMENDED READING: "Robbins Basic Pathology" 11th Edition, 2023. Chapter 5: Diseases of Immune System (pg. 130-185). Cells of the Immune System, pg. 133-137; Overview of Lymphocyte Activation and Adaptive Immune Response, pg. 138-140; Hypersensitivity: Immunologically Mediated Tissue Injury, pg. 141-149; Autoimmune Diseases, pg. 149-160; Immunodeficiency Syndromes, pg. 165-170. PART I BIOLOGY OF THE IMMUNE SYSTEM Introduction Definitions: The immune system is a collection of mechanisms that protects against disease by identifying and killing pathogens, and tumour cells, and protection against microbial toxins. Immunology is the science that examines the structure and function of the immune system. PATHOLOGY 3500/PATHOLOGY 9535 1 IMMUNITY AND IMMUNE DISORDERS Pathogens: Include: viruses, bacteria, mycobacteria, parasites, and fungi. ↳ harmful + not native to tody/system GENERAL CHARACTERISTICS OF IMMUNITY RSRAM ↳ Real Shit RAM Q ② 1. Recognition: The ability to distinguish between normal self, altered (damaged) self and ③non-self (foreign material). 2. Specificity: The ability to inactivate, destroy and remove the "offending" material, without damaging normal tissues in the vicinity of the reaction, i.e. the reaction must be target-specific. - 3. Regulation: The immune system is able to control the00 type, intensity and C duration of the reaction and has the ability to prevent immune reaction (suppression). 4. Amplification: The effector (attack) phase of the immune reaction is mediated through multiple pathways which act synergistically for optimal effect. Each pathway has built-in amplification systems, too. All these systems have different triggering points and each may be triggered independently, but eventually involve the other systems. 5. Memory: The identity of the foreign material (antigen) which led to the first (primary) immune response is remembered so that the next episode involving the same antigen will result in an accelerated reaction (secondary immune response), which by-pass several initial steps that the primary immune response has to go through. Immunological memory is what confers - long-term immunity against infections. - Is 120 responses KEY FEATURES OF THE IMMUNE SYSTEM - It ↳ innate I acquired Defense against microbial invasion involves two types of systems: innate and acquired (adaptive) immunity. Our innate immune system does not require prior exposure to a microbe to mount an immune response – it is always present and ready to attack. Acquired immunity is a more advanced system requiring exposure to an antigen in order to become active against microbes that have evaded the innate system. CHARACTERISTICS OF THE INNATE (NATURAL) IMMUNE SYSTEM: Go to first normal response - 1. Exposure leads to immediate maximal response. 2. It is non-specific. 3. It does not require a previous exposure to an offending agent (antigen). 4. Found in nearly all forms of life CHARACTERISTICS OF THE ADAPTIVE (ACQUIRED) IMMUNITY: how it reacts knowing how - to deal w/the problem 1. Pathogen and antigen-specific response. 2. Lag time between exposure and maximal response. Assess how to react 3. Cell-mediated and humoral (antibody) components. 4. Cell-mediated and humoral components (of inflammatory response). 5. Exposure leads to immunological memory. 6. Found only in jawed vertebrates. ↳ due to evolutionary development vertebrates ↳ specialized lymphocytes only present - in jawed INNATE IMMUNITY (covered in Robbins, Chapter 2, also review notes on acute inflammation) THE INNATE IMMUNE RESPONSE takes place when a microorganism is able to break through the normal epithelial barriers of the skin, GI and respiratory tract. PhagocytesO - ingest microbes ↳ All the linnings PATHOLOGY 3500/PATHOLOGY 9535 2 IMMUNITY AND IMMUNE DISORDERS  and E secrete cytokines which stimulate the inflammatory response. Cells have various receptors (pattern recognition receptor) that are able to recognize components that are preserved among broad groups of microorganisms. COMPONENTS OF THE INNATE IMMUNITY: Sea Horse 1. Surface barriers a. Mechanical, such as skin b. Chemical, such as enzymes in saliva, vaginal secretions and tears 3 mucosal linnings c. Biological, such as bacterial flora in different organs 2. Humoral and chemical barriers: a. Inflammation: is one of the first responses of the immune system to infection. It is produced as a result of release of: o Cytokines (such as interleukins) released by infected or injured cells o Prostaglandins group of lipid compounds derived from arachidonic acid [fatty acid) local signalling molecules - - Conj double donds o Leukotrienes inflammatory mediators derived from AA*- regulate immune responses. , bonds,- - - o Chemokines regulate the migration positioning of immune cells during both homeostasis & inflammation 3 conj. double - O o Interferons immune response to viral infections regulation of - activity & immune The cellular components of inflammation are: o Neutrophils: phagocyte and release enzymes. o Eosinophils and basophils: secrete chemical mediators. o Monocytes/macrophages: attack pathogens by engulfing and then killing the microorganism by enzymes present within “lysosomes”. o Mast cells: regulate the inflammatory response. o Dendritic cells: phagocyte. o Natural killer cells. "killing system" b. Complement system: it consists of more than 20 proteins and named as such due its ability to “complement” the killing of a pathogen. They are synthesized - mainly in the liver and normally circulate in the blood in inactive form. The complement proteins can be activated by: o Proteases (damaged cells, bacterial endotoxins), or o Binding of the complement to antibodies that are attached to microbes, or - o Binding complement to carbohydrates on the microbes’ surfaces. - Jasically > - , when microses are binded to antibodies or carbohydrates The complement activation results in: ▪ Cell membrane disruption (lysis of target cell), or ▪ Opsonization (coat) an organism, marking it for destruction, or ▪ Attraction of other immune cells through production of peptides. body's immediate & ▪ Complement activation also results in the release of various factors, e.g.- early response to ① peptide fragments 232 C42 152 Q ② anaphylatoxins and chemotactic factors which result in acute inflammation. injury/infection - 2 , , migration of the towards - ② direct the cells ▪ Certain products of complement activation can also trigger the coagulation a reas of infections system, kinin system and fibrinolytic system. Invasion by microbes usually occurs across the main epithelial barriers. Epithelia are a physical underneath the barrier to entry. Once across the epithelium, microbes face attack by phagocytes, including epithelial tissue - macrophages that reside within the subepithelial tissues, and neutrophils which are rapidly - - located in Oval recruited to the site (see inflammation notes). Phagocytes recognize microbes through Cavity evolutionarily conserved receptors, especially Toll-like receptors (TLRs). These are a family of ‘pattern-recognition’ receptors which recognize products of bacteria (endotoxin, etc), viruses (double stranded RNA), and other pathogens (see fig. 2-3, pg 32). The phagocytes kill microbes by ingesting them (phagocytosis) and production of microbicidal substances. Phagocytes (and dendritic cells) also produce cytokines which enhance killing of microbes and recruitment and activation of other cells of the immune system. - Major Histo compatibility Complex Natural killer cells recognize class I MHC molecules (see MHC section below), which are - PATHOLOGY 3500/PATHOLOGY 9535 3 IMMUNITY AND IMMUNE DISORDERS Subtype Nucleus Function Example Bacterial fungal infection. Most ⑳ or common Multi-lobed responders to microbial infection Neutrophil Parasitic infections & reactions · allergic (inflammatory) Eosinophil Bi-lobed Allergic & antigen response (releases Basophil Bi/Tri-lobed histamine causing vasodilation) D Include B cells , CD2 t helper T cells & + CD8 ⑳ Deep staining, , Cytotoxic T cells · Operate primarily in the Lymphocyte Ecentric lymphatic system Phagocytosis of pathogens. Presentation of antigens Kidney snaped Eventually , they ⑭ to T cells. become tissue macrophages, Monocyte which remove dead cell debris & attack microorganisms present on all healthy cells. NK cells express inhibitory and activating receptors. The receptor for MHC class I is an inhibitory receptor, therefore NK cells will be ‘inhibited’ from attacking normal healthy cells. However if a cell is damaged or abnormal (i.e. virally-infected cell, tumour cell), such that MHC I is abnormal or not expressed, NK cells will kill them. In addition, damaged or stressed cell may express molecules that bind to the activating receptors on NK cells. In the absence of normal MHC I, NK cells will become activated to kill these cells. NK cells also function as part of the adaptive immune system by recognizing antibody coated cells, which they will also kill (antibody-mediated cytotoxicity, see below). NK cells also produce the cytokine interferon- in order to activate macrophages. Some plasma proteins, particularly the complement system, recognize components of microbes (endotoxin, mannose residues) and are activated. The complement system consists -proteins of a group broken of proteins that are present in plasma in inactive form. Once activated via proteolysis they may down into smaller polypeptides/At - form complexes with other complement proteins to kill microbes by direct cell lysis (membrane attack complex). They may also act as inflammatory mediators to recruit leukocytes, or may act as opsonins (C3b), coating microbes to target them for phagocytosis (see Robbins Fig. 3.11). ACQUIRED (ADAPTIVE) IMMUNITY Adaptive immunity is dependent on several cell types (lymphocytes, antigen presenting cells, some phagocytes). Specificity is achieved through recognition of specific antigens and expression of MHC molecules on particular cell types. It allows for stronger immune responses and immunological memory, and requires the recognition of a specific foreign (non-self) antigen. developing lymphocytes /B occurs in cells & + cells) to generate j antibodies the diverse & T-cell receptors forrecognizing antigens ↑ Variable , Diversity , joining of gene segments of immunoglobulin genes The system is highly malleable due to the somatic hypermutation and V(D)J recombination of antigen receptor genes. This process allows a small number of genes to generate enormous number of antigen receptors that are uniquely expressed on each individual lymphocyte. Components of adaptive immunity: Lymphocytes 3 all hold up o T lymphocytes memory o B lymphocytes o Natural Killer (NK) cells (see above) Other cells: o antigen presenting cells ▪ dendritic cells ▪ macrophages o phagocytes ▪ macrophages Human major histocompatibility complex (MHC) ↳ proteins found on the surface of all nucleated cells intracellular peptides Cytotoxic T Cells ((D8 + cells) ↳ present (from viruses or abnormal proteins) to The major functions of the adaptive immune system include: o The recognition of specific “non-self” antigens during the process of antigen presentation. o The generation of responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells. PATHOLOGY 3500/PATHOLOGY 9535 4 IMMUNITY AND IMMUNE DISORDERS  o The development of an immunologic memory, in which a signature antigen in each pathogen is “remembered” or “recognized”. These memory cells can be recruited to quickly eliminate a pathogen if a subsequent infection occurs. T-LYMPHOCYTES subset of white Good cells-leukocytes They originate from primitive stem cells (yolk sac in embryos and bone marrow after birth), and mature in the thymus gland. They constitute 60 to 70% of peripheral blood lymphocytes. Each > - - cell is programmed to recognize a specific cell-bound antigen by means of an antigen-specific T- Cell Receptor (TCR). TCRs are linked to a cluster of five polypeptide chains, called CD3 molecular complex. CD3 molecules do not bind antigen but are involved in the transduction of signals into the T cell after it has bound the antigen. T-lymphocytes also express a variety other molecules including CD4 or CD8. [CD=cluster of differentiation] CD4 (expressed on ~60% of mature CD3+cells) and CD8 (expressed on ~30% of T cells) are very important. They provide the Helper/inducer and suppressor/cytotoxic functions respectively. Antigens are presented to T-cells by accessory cells (antigen presenting cells) that carry an appropriate histocompatibility (MHC) molecule. 0 O 8 = 2 = 4 - - Class CD4 8 = whole L = ↳ (D8 = class 1 j C C Similar but note the differences ------------------------------------------------ B-LYMPHOCYTES Constitute 10-20% of peripheral lymphocytes. Arise from yolk sac in embryos, and bone marrow after birth. They mature in the Bone marrow. Immature B-cells (pre-B) contain cytoplasmic heavy-chain immunoglobulins (Ig). Later, they develop surface immunoglobulins (Ig). Mature B-cells are primarily in a resting state, awaiting activation by foreign antigen. MADGE On antigenic stimulation, they form plasma cells which secrete 5 classes of immunoglobulins GAMED (M, G, A, D, E). Like the T lymphocytes, B-cells recognize antigen via the B-cell antigen BLR > - DAMGE receptor complex. The major one is IgM antigen receptor complex. Other receptors are Y complement receptors, IgA and IgE, CD40 and Fc receptors. pronounced "Damage" ↳ s bind to specialized Fo regions PATHOLOGY 3500/PATHOLOGY 9535 5 IMMUNITY AND IMMUNE DISORDERS  B Lymphocytes - have DAMGE Cimmunoglobulins) CD4 OCD8 T-lymphocytes have - O O O Of I immens me sam T-cells and other non-specific factors (e.g. bacterial products and certain factors) are required for maturation and differentiation of B-lymphocytes. MACROPHAGES They are phagocytic cells, present virtually in all body organs. They are required to: - Process and present antigen to immunocompetent T-cells. Important in certain cell-mediated immunity such as delayed hypersensitivity reaction Important in the effector phase of humoral immunity (phagocytose opsonized microbes). They secrete macrophage-derived cytokines which amplify T-cell responses. DENDRITIC CELLS Antigen presenting cells: Interdigitating dendritic cells, expresses high levels of major histocompatibility complex (MHC) antigens Follicular dendritic cells, bear Fc receptors for IgG - ↳ constant part of an antibody molecule NATURAL KILLER CELLS that interacts w/ immune cells 10-15% of peripheral blood cells; do not bear T-cell receptors or cell surface immunoglobulins They have innate ability to lyse a variety of tumour cells, viral infected cells and some normal cells without previous sensitization. Ability to lyse IgG-coated target cells (antibody dependent cell-mediated cytotoxicity [ADCC] They have CD16 and CD56 surface molecules Interferon promotes their killing activity and prostaglandin E2 is highly suppressive of NK cells. HUMAN MAJOR HISTOCOMPATIBILITY COMPLEX The MHC is an intricate system of membrane proteins or antigens, referred to as human leukocyte antigens (HLA). MHC genes are located on chromosome 6 and code for three major classes of molecules (designated as I, II & III). Class III is complement antigen and NOT a histocompatibility antigen. Class I molecule is present on all nucleated cells and recognized by cytotoxic T-cells. Class II is limited to: Antigen presenting cells B-cells Subsets of activated T cells PATHOLOGY 3500/PATHOLOGY 9535 6 IMMUNITY AND IMMUNE DISORDERS  Feature Complement Antigen Histocompatible Antigen System Involved Innate Immune System Adaptive Immune System complement proteins ↑ cell of peptides bound receptor recognition Recognition Mechanism ex. (3b binding to surfaces to MHC self/non-self Pathogen-associated molecular patterns MHC molecules presenting peptides Target CPAMPs) , immune complexes Ennances a Determines compatibility in transplants & Rolf pathogen clearance immune inflammation recognition Bacterial or viral antigens activating MLA molecules determining tissue graft Examples complement pathways compatibility  SPECIFIC FUNCTIONS OF THE ACQUIRED IMMUNE SYSTEM ANTIGEN PRESENTATION Antigen presenting cells (APCs) reside in tissues where one of their major roles is surveillance against microbial invasion. Microbes that gain entry through epithelial surfaces are captured by dendritic cells. Dendritic cells ingest the microbe or proteins from the microbe, then proteolytically digest them such that peptide antigens are produced. These “extracellularly- derived” peptides are then ‘presented’ on the surface of the dendritic cell bound to MHC class II molecules. Dendritic cells will migrate through the lymphatic system to downstream lymph nodes. Once there, the presented antigens are recognized by CD4+ helper T-cells. Since MHC class I molecules are present on all cells, dendritic cells also express MHC class I and can present intracellular antigens (i.e. those derived from intracellular pathogens like viruses) to CD8+ cytotoxic T-cells (Robbins Fig. 5.9). CELL-MEDIATED IMMUNITY CD4+ helper T-cell response: The main function of CD4+ T-cells is to ‘help’ activate other cell types involved in immunity. This helper function is mediated through production of cytokines and through expression of CD40 ligand (CD40L). Once activated via recognition of specific antigen presented by APCs, CD4+ T-cells secrete the cytokine interleukin-2 (IL-2), and also CD4 express a high-affinity receptor for IL-2. IL-2 induces proliferation of T-cells, allowing for expansion of an antigen-specific T-cell population. Some of these activated T-cells differentiate into specific types of effector T-cells which perform specific functions; these include T H1 cells Mary · IFN- y which secrete IFN-, TH2 cells which secrete IL-4, IL-5, and IL-13, and TH17 cells which secrete IL-17.①IFN- activates phagocytosis and production of antimicrobial substances in Th2 macrophages, as well as antibody production in B-cells.②TH2 cells are mainly involved in · T+17 activation of mast cells and eosinophils, rather than macrophages.⑤TH17 cells are involved in · CD4OL activation of neutrophils.⑪CD40L is expressed on the surface of CD4+ T-cells, and binds to · CD40 on B-cells and macrophages to help activate them in conjuction with the secreted cytokines. specialize in identifying ↑ A killing infected , cancerous, CD8+ cytotoxic T-cell response: once activated the CD8+ T-cells differentiate into cytotoxic 26 normal cells - lymphocytes. These cells function to destroy cells infected by microbes, often viruses or - CDG intracellular bacteria, in order to eliminate the infection. Recognition of infected cells occurs via MHC class I molecules: the infected cell can present peptide antigen derived from the forms pores in the infectious organism bound to MHC I. CD8+ cells specific for that antigen will bind antigen ~ and membranes of target MHC class I, then destroy the cell. CD8+ cytotoxic cells produce the proteins perforin and 2 cells - 2 induces a poptosis in granzyme-B which are stored in cytoplasmic granules. These proteins are released into target - ↳ destroys them targetcells cells (perforin allows entry of granzyme-B across plasma membrane) whereby granzyme-B ex virus infected or triggers apoptosis via proteolytic cleavage and activation of caspases (family of proteases which , lancerous - activate apoptotic death). ↳ clears protein HUMORAL IMMUNITY Once activated, B-cells differentiate into plasma cells or memory B-cells. Activation can occur in a T-cell-independent or –dependent manner. T-cell-independent activation occurs with polysaccharide or lipid antigens, which may have multiple identical epitopes that can bind to several B-cell antigen receptors. T-cell-dependent activation occurs with protein antigens which cannot bind to many antigen receptors, therefore full activation requires T-cell mediated cytokine stimulation coupled with CD40 ligand expression (co-activation). ↳ CDU + presents on cells Plasma cells are antibody secreting cells. When a specific B-cell becomes activated it then undergoes proliferation/clonal expansion. Some of these clonal cells differentiate into plasma cells which secrete antibodies with identical antigen-specificity to the B-cell receptor that first PATHOLOGY 3500/PATHOLOGY 9535 7 IMMUNITY AND IMMUNE DISORDERS recognized the antigen. Secreted antibodies have several functions including binding to antigen on microbes in order to neutralize them, activation of the complement system, and opsonization (coating) pathogens to target them for phagocytosis. There are 5 classes of antibodies: DAMEG IgG – opsonize microbes, activates complement, crosses the placenta (passive immunity) G for great > - IgM – activates complement M for ment complement - - responsibility IgA – secreted in mucosal tissues (protection of mucosal epithelia) A : 1 Secretes first line of defense mucosal - > > - IgE - coats certain parasites, activates mast cells and eosinophils E En coats parasites ~ : > - tissues IgD – specific functions uncertain Some cells of the immune system such as neutrophils, macrophages and natural killer cells have cell surface receptors to the Fc portion (non-variable) of antibodies, allowing them to recognize and destroy (in a non-specific manner) opsonized cells/microbes. This is known as antibody dependent cell-mediated cytotoxicity (ADCC). Memory B-cells and T-cells are long-lived effector lymphocytes whose purpose is to allow fast activation upon exposure to the specific antigen they recognize. :. adaptive in immunity specializes g ↑ Differentiation 8:2 = 4 C MHC2 > - CD4 PART II IMMUNE DISORDERS Disorders of the immune system may be broadly divided into: i. Hyperfunction too much function - ii. Hypofunction -not enough function In general, hypofunction or immunodeficiency results in two main forms of disease, as you would expect if you recall the two basic functions of the immune system: ① Defense PATHOLOGY 3500/PATHOLOGY 9535 8 IMMUNITY AND IMMUNE DISORDERS  ②Surveillance Disorders of defense, the most common manifestation of immune hypofunction, results in increased susceptibility to infections. The type of infection seen depends on whether cell-mediated, humoral or both forms of immunity are affected. ↳ ex immunocompromised individuals but not > -> necessarily Disorders of surveillance lead to increased frequency of malignant disease. Patients with immunodeficiency syndromes and those on immunosuppressive medications have a much higher incidence of cancer. Hyperfunction, usually termed HYPERSENSITIVITY, results in damage of normal tissue. We shall now look at hypofunction and hyperfunction of the immune system in greater detail and examine specific examples. HYPERSENSITIVITY REACTIONS Hypersensitivity reactions are those reactions causing tissue injury. These often represent “excessive” immune response to an antigen. Hypersensitivity reactions may result from immune response to self antigens (autoimmunity – see below), reactions to microbes, or reaction to environmental antigens. The disease states that result from hypersentitivity reactions are often chronic due to persistence of the antigens and amplification of the immune response. There are 4 types of immune reactions which may lead to tissue damage or disease: TYPE I: Anaphylactic tone act blood vessels altering their , IgE/mast cell interaction. - permeability on , A diameter. Release of vasoactive amines. (e.g. histamine) - Examples: Asthma, hayfever, bee sting, peanuts. poly unsaturated fatty acid In type I reactions result from TH2 helper T-cells, which help in the activation of IgE-producing B- cells/plasma cells. IgE molecules are attached to mastv cells, which become activated and ↑ degranulate (release histamine, leukotrienes, prostaglandins and other mediators) when IgE - binds antigen in a previously sensitized individual. This are termed allergic - (or atopic) reactions. immune responses to The effects of the reaction include vascular permeability and smooth muscle contraction. The environmental mediatedby type allergens 1 reaction can be localized or systemic. Depending on the severity, the reaction may be life- hypersensitivity threatening (i.e. anaphylactic reaction). - 4 systemic - involve multiple organ systems driven by a massive release of inflammatory mediators like Histamine from mast cells A Jasophils through IgE-mediated mechanism TYPE II: Antibody-mediated - IgG or IgM/complement interaction. Lysis of cells. immune ↑ w Example: Transfusion reactions due to mismatched blood, thrombocytopenic purpura (ITP), - formation Goodpasture syndrome. ↳ low platelet count that leads to increased bleedinga of purpura ismal people bruises under the skin) In type II reactions antibodies are produced which bind to antigens on surfaces of cells or tissue components. These antibodies can then activate complement, or bind to Fc receptors on phagocytes. Thus, circulating cells which are opsonized by the antibody may then be targeted for destruction by phagocytes (e.g. ITP), while other cells and tissues targeted by the antibodies are damaged secondary to complement activation and subsequent inflammation. TYPE III: Immune complexes Tissue damage caused by the overactivation/ dysregulation of the complement system Antigen-antibody complexes in the M circulation are trapped in various organs (e.g. kidney) ~ where they produce injury by - complement activation and neutrophil activation. Example: Systemic lupus erythematosus, post-streptococcal glomerulonephritis. A Streptococus ↳Chronic autoimmune disease - production of ↳ Kidney disease that occurs following an infection u/group autoantibodies infections against self-antigens > - widespread causing pharyngitis or skin inflammation & tissue damage across multiple organ systems is characterized by inflammation of the glomeruli > - acute nephritic syndrome PATHOLOGY 3500/PATHOLOGY 9535 9 IMMUNITY AND IMMUNE DISORDERS  In type III reactions antibodies are directed against circulating antigens. These antigens may be endogenous (e.g. nucleoproteins) or exogenous (e.g. proteins from microbes). The antibodies and antigens bind together to form circulating immune complexes. Immune complexes form in the course of normal immune responses; it is only when formed in large amounts that they cause problems. These are eventually deposited in tissues where they activate complement and neutrophil-mediated inflammation. The complexes may be deposited in blood vessels, including blood vessels of specific organs, or several tissues/organs (systemic). The tissue damage is manifested by necrotizing vasculitis (acute inflammation within the blood vessel wall with - necrosis), associated ↳ischemic necrosis and acute inflammation in the affected surrounding necrosis of flood vessel walls tissue. TYPE IV: (Cell-mediated immunity) Two types: ① delayed-type hypersensitivity mediated by CD4+ T-cells o examples: contact dermatitis due to poison ivy, tuberculin reaction, granulomatous - inflammation TB test ↳) ② T-cell-mediated cytotoxicity, mediated by CD8+ T-cells o examples: viral hepatitis, type I diabetes, solid-organ transplant rejection In delayed-type hypersensitivity, CD4+ T-cells differentiate into different classes of helper T-cells upon exposure to antigen via antigen-presenting cells. Depending on the particular cytokine(s) secreted by the APC, the T-cell differentiates into either TH1 or TH17 cells. Upon subsequent exposure, the differentiated T-cells migrate to the site of the antigen and are activated in conjunction with APCs. The TH1 cells secrete IFN-, a cytokine which activates macrophages, promoting phagocytosis and production of microbicidal - substances. T H17 secrete IL-17, a producedbyimmeeaster cytokine which is chemotactic for neutrophils, recruiting them to the site of exposure and inciting externally to kill/inhibit acute inflammation. In cases of delayed-type hypersensitivity, tissue damage results. Formation the growth of microbes Such as bacteria viruses, of granulomas (def: aggregate of epithelioid macrophages surrounded by a rim of lymphocytes – , fungi , o parasites see Robbins Fig. 5.18) occurs in some types of delayed-type hypersensitivity where there is persistence of an organism or other antigen (e.g. tuberculosis). The initial CD4+ T-cell infiltrate is replaced by activated macrophages which fuse to form multinucleated giant cells. In T-cell mediated cytotoxicity CD8+ cells destroy cells expressing a particular antigen (eg. self antigen expressed on islet cells of the pancreas in type I diabetes). PART III – AUTOIMMUNITY AND IMMUNODEFICIENCY AUTOIMMUNE DISEASE Autoimmune diseases occur due to a breakdown of the normal processes which maintain a state of immunological tolerance to self-antigens. The current concepts of immunological tolerance involve two main factors: Discrimination of self and non-self antigens by antigen reaction T cells (recognition). Suppression of immune responses to self antigens by suppressor T cells. It is thought that autoimmunity results from two main factors: ~ mone system mistakenly targets & Itracks the body's own tissues ① Genetic susceptibility o autoimmunity runs in families o many patients have more than one autoimmune disease - Antigen--G Human Cerkocyte o particular HLA alleles are linked to autoimmune diseases o genetic polymorphisms are linked to autoimmune diseases PATHOLOGY 3500/PATHOLOGY 9535 10 IMMUNITY AND IMMUNE DISORDERS  ②Environmental factors o microbes including viruses and bacteria may trigger autoimmune diseases o UV radiation o ?smoking o tissue damage o ?hormones The clinical picture in the different autoimmune diseases depends on: a) the target (antigen). b) type of immune reaction (cell-mediated, humoral or both). c) changes secondary to the destruction of the target organ or type of immune reaction. Examples: 1. Systemic lupus erythematosus: a) Target: DNA b) Immune reaction: Type III - circulating DNA-antiDNA complexes c) Dermatitis, nephritis, arthritis: Due to trapping of complexes in skin, kidneys and joint synovium. 2. Hashimoto's thyroiditis: a) Target: thyroid follicular cells b) Immune reaction: Type II - cytotoxic antibody - complement activation Type IV - cell-mediated c) Hypothyroidism due to destruction of thyroid cells. IMMUNODEFICIENCY DISEASES Primary (congenital) Secondary (acquired) Clinical Features Associated with Immunodeficiency 1. Chronic infection. 2. Recurrent infection (greater frequency than expected). 5 ex Mama's constant. painsa swelling 3. Unusual infecting agents (low pathogenic potential). 4. Poor resolution or poor response to antibiotic treatment. 3 can't be treated w/pills Primary Immunodeficiency Diseases: Certain generalizations may be made regarding immunodeficiency syndromes: Cell-mediated immune deficiencies (T-cell dysfunction) may present very early (such as during the neonatal period) (e.g. DiGeorge syndrome - congenital absence of thymus). It usually affects the humoral system as well because of a lack of helper/suppressor effect on B cells. Thus, pure T cell dysfunction is unlikely Pure B cell dysfunction (with normal T cell function) is possible (e.g. Bruton's syndrome). Pure B cell dysfunction is not detected until the infant is 5-6 months old, because of protection by maternal IgG antibodies. Both T and B cell deficiency can occur in the same patient (e.g. severe combined immunodeficiency disease). PATHOLOGY 3500/PATHOLOGY 9535 11 IMMUNITY AND IMMUNE DISORDERS  at birth present Specific primary (congenital) immunodeficiency disorders (refer to Robbins for details on the specific disorders listed below) Disorders affecting lymphocyte function X-linked agammaglobulinemia lack of mature B cells leading to reduced levels of immunoglobulins - , Common variable immunodeficiency defect in antibody production > recument infections - - Isolated IgA deficiency inability to produce adequate levels of IgA - Hyper-IgM syndrome too much IgM - DiGeorge syndrome -chromosome deletion abnormalities in multiple organ systems > - Severe combined immunodeficiency life disease caused by defects in both T & B-cells threathing - Recurrent infections may also occur in the presence of intact T and B cell function. These are due to defects in the amplification systems, e.g. deficiency of specific complement components, disorders of the mononuclear phagocytic system and of the granulocyte series. Chronic granulomatous disease results from phagocytic dysfunction (absence of lysosomal enzymes in monocytes and granulocytes). Disorders affecting innate immunity Deficiency of complement proteins Chronic granulomatous disease (defect in NADPH oxidase) Rare mutations in Toll-like receptors - ↳ class of proteins that help the body's innate immune system recognize& respond to microbial antigens Secondary Immunodeficiency Diseases: ↳ Acquired conditions resulting in impaired immune function > - Acquired later in life These may occur due to: 1. Infections: Rubella } Temporary Measles }----- Immuno- Mycoplasma } deficiency Human Immunodeficiency Virus (HIV) → Acquired Immunodeficiency Syndrome (AIDS) 2. Immunosuppressive therapy: i. Cytotoxic drugs, cortisone (e.g. chemotherapy for cancer) ii. Irradiation iii. Anti-lymphocyte serum globulin (ALG) 3. Malignancy (especially lymphoma) 4. Chronic illness 5. Malnutrition 6. Aging PATHOLOGY 3500/PATHOLOGY 9535 12 IMMUNITY AND IMMUNE DISORDERS ③ MOLECULAR GENETICS DR. L. SCHENKEL PATHOLOGY AND LABORATORY MEDICINE, LHSC, WESTERN UNIVERSITY OBJECTIVES: 1. Define the different types of gene mutations 2. Understand the molecular basis of diseases: a. Single gene x Polygenic (multifactorial) x Epigenetic x Mitochondrial 3. Describe and provide examples of Mendelian inherited diseases. 4. Describe Non-Mendelian (atypical) inheritance and provide examples of disorders with a) trinucleotide repeat expansions b) mitochondrial inheritance c) genomic imprinting Recommended readings: "Robbins Basic Pathology" 11th Edition, 2023. Chapter 4 (pg. 79-230) - read relevant sections; basic genetics textbooks (e.g. Lewin’s Gene X). TYPES OF GENETIC MUTATIONS: Mutations are permanent changes in DNA. o Single nucleotide variants (SNV): missense, synonymous, nonsense o Insertion and deletions: inframe or frameshift o Copy number variants (CNV): >500 bp to several Kb o Dynamic mutations: trinucleotide expansions Significance of gene mutations: o Somatic (mosaic) and germ-line mutations o Heterozygous, homozygous, hemizygous o Gain of function and loss of function mutations o Complete dominance: heterozygous and homozygous for the dominant allele have the same phenotype o Incomplete dominance: the phenotype of the heterozygous falls between the phenotype of the homozygous (WT and mutant) o Codominance: expression of both alleles in heterozygous state (example ABO group) o Haploinsuficiency: a single copy of the gene is not sufficient to produce the wild-type phenotype o Hypomorphic: loss of function mutation that reduce gene activity o Amorphic: mutation eliminates gene activity MOLECULAR BASIS OF DISEASES SINGLE GENE DISORDERS: Determined by the allele at single locus (single gene) and follow the classic (Mendelian) inheritance patterns in families (autosomal recessive, autosomal dominant and X-linked). 2 Gregor Mendel laws: Law of dominance Some alleles are dominant while others are recessive; an organism Q and uniformity with at least one dominant allele will display the effect of the dominant allele. PATHOLOGY 3500/PATHOLOGY 9535 1 MOLECULAR GENETICS  Law of segregation During gamete formation, the alleles for each gene segregate from ② each other so that each gamete carries only one allele for each gene. Law of independent Genes of different traits can segregate independently during the ③ assortment formation of gametes. Mendelian inheritance 1. Autosomal recessive – usually both parents are carriers of the mutant gene but are not clinically affected; disease occurs in homozygous or compound heterozygous state (two alleles relationship Jetween with mutation); When both parents are carrier, each child has a 25% chance of being affected; individuals - who are both males and females are affected; complete penetrance is common; consanguinity is a from a Incestor common - strong evidence for AR; new mutations are rarely detected clinically; commonly seen in metabolic conditions - enzyme proteins are affected by the mutation. Diseases include cystic fibrosis, phenylketonuria, galactosemia, Tay-Sachs disease, mucopolysaccharidoses. 2. Autosomal dominant – usually one parent is affected; both males and females are affected, either can transmit the condition; disease occurs in heterozygous state (one allele with mutation); new mutations (de novo) are detected clinically; any child of affected parent has 50% chance of inheriting the disease; clinical features can be modified by reduced penetrance and variable expressivity; regulatory (i.e. receptor) and structural proteins not enzymes are affected by the mutation. Diseases include: Familial Hypercholesterolemia, Marfan Syndrome, Ehler-Danlos Syndrome. 3. X-linked recessive – incidence of trait is higher in males (hemizygous) than females; heterozygous females are often unaffected but may express the condition due to X-inactivation or homozygosity; mutant gene is transmitted from affected males to all daughters but not sons; for a heterozygous female, each son has a 50% chance of being affected and each daughter has a 50% chance of being a heterozygous carrier. Disease include Hemophilia A. 4. X-linked dominant – females heterozygous are affected; lack of father-son transmission; may result in hemizygous male lethality. Disease include Rett syndrome. Pedigrees: graphical representation of the family tree used to summarize family history and establish the pattern of transmission PATHOLOGY 3500/PATHOLOGY 9535 2 MOLECULAR GENETICS · in every generation not present every < both genders generation affected XX XY Dad -> his X-chromosome XY has the issue &Daughters XX XX XX XX XY XY XY - MULTIFACTORIAL (COMPLEX) DISORDERS Multifactorial diseases with complex inheritance result from interaction of multiple genetic variants and environmental factors. Threshold model: At a point an individual accumulates a certain liability that will be affected by the disorder. The level of liability at which this occur is referred as threshold level 2 At this point , disease (insert Situation] will be expressed Disease examples: congenital malformations, diabetes, schizophrenia, asthma, Alzheimer. EPIGENETIC DISORDERS Epigenetic is defined by modifications of DNA and its associated proteins (histones) that do not involve alterations in the DNA sequence. They are responsible for controlling gene expression, specification of cell types, X-inactivation, Imprinting. Types of epigenetic modifications: o DNA methylation o Histone modification o Non-coding RNA o Histone variants Genomic imprinting: refers to parent-of-origin-specific gene expression. For a significant proportion of our genes, only one of the two alleles is expressed—the other allele is silenced. The repression of one allele is mediated by epigenetic factors. PATHOLOGY 3500/PATHOLOGY 9535 3 MOLECULAR GENETICS  Maternal imprinting refers to silencing of maternal allele and paternal imprinting to silencing of paternal allele Imprint is reset during gametogenesis and is stably transmitted to all somatic cells derived from zygote For imprinted genes, only one functional copy exists in the individual so loss of the functional allele leads to disease Examples of diseases caused by aberrant imprinting: Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are best examples of genomic imprinting disorders. PWS and AS result from disruption of genes on the paternally inherited and maternally derived chromosomal region, respectively. The disruption can result from deletion (up to several megabases of DNA), uniparental inheritance (both chromosomes from one parent), abnormal methylation imprint. AS also results from mutation in UBE3A ligase gene which is paternally imprinted (i.e. expressed from maternal allele). TRIPLET REPEAT EXPANSION It is defined as an unstable expansion of repeating units of 3 or more nucleotides that occur in tandem. It is also known as dynamic mutation as the repeat size can change from generation to generation (expand during gametogenesis). o Follow non-Mendelian pattern of inheritance o Trinucleotide expansion may involve any part of the gene (introns, exons, untranslated regions); often include CGs – for example, CGGs in Fragile X Syndrome and CAGs in Huntington’s disease o Result in loss of protein function (mutation in 5’ untranslated region: Fragile X Mental Retardation) or gain of (toxic) function (mutation in coding region – abnormal protein: Huntington’s disease) o Mutation is dynamic. When a certain threshold is met for ‘normal number’ of triplet repeats (referred to as premutation alleles) then trinucleotide repeat expansion occurs during gametogenesis and leads to a full mutation. Threshold for converting a premutation to full mutation differs with each disorder; amplification occurs in either oogenesis (eg. Fragile X syndrome) or spermatogenesis (eg. Huntington’s disease) depending upon disorder. Fragile X syndrome FXS is the most common inherited cause of intellectual disability in males. It is caused by trinucleotide repeat (CGG) expansion in the 5’UTR of FMR1 gene associated with DNA hyper- methylation of gene promoter and repression of gene expression. Fragile X syndrome differs from classical X-linked recessive inheritance in that: some males can carry the “premutation” repeat allele and pass this to unaffected daughters who then transmit an expanded repeat to their sons who are affected. These differences are attributed to the dynamic nature of the CGG expansion. In the normal population, less than 50 CGG repeats are present whereas the full mutation has >200 CGG repeats. The full mutation arises through a premutation stage (59-200 CGG repeats). Both males and females (ie. carriers) have premutations but they can only be expanded to full mutations during oogenesis. These expansions can in turn be transmitted to either sons or daughters of the carrier female. - Premutations can also have a direct clinical effect on carriers. About 1/3 of female carriers have premature ovarian failure and ~1/3 of male premutation carriers develop Fragile X associated tremor/ataxia syndrome later in life. MITOCHONDRIAL DISORDERS mtDNA: small circular, double strand DNA. Contain 37 genes (13 proteins coding genes and rRNA and tRNAs. High mutation rate. >1000 mtDNA per cell Mitochondrial DNA encode enzymes involved in oxidative phosphorylation. PATHOLOGY 3500/PATHOLOGY 9535 4 MOLECULAR GENETICS Maternal inheritance: Zygotes have only maternally derived mitochondria. Mothers transmit mitochondria to all their offspring (sons and daughters) but only daughters transmit mitochondrial DNA to their offspring. Follow non-Mendelian pattern of inheritance. Homoplasmy: pure population of mtDNA Heteroplasmy: mixed or normal and mutant mtDNA Mitochondrial diseases caused by mutation in mtDNA genes: typically involve neurodegenerative, cardiovascular, neurometabolic, visual, auditory and muscular disorders. Examples: Leber Hereditary Optic Neuropathy (LHON), Myoclonic epilepsy and ragged red fibers (MERRF). Note that mitochondrial diseases can also be caused by nuclear encoded genes involved in oxidative phosphorylation. This will follow Mendelian pattern. PATHOLOGY 3500/PATHOLOGY 9535 5 MOLECULAR GENETICS ④ MOLECULAR GENETICS TECHNIQUES and GENOMIC APPLICATIONS IN NEOPLASIA DR. E. LALONDE PATHOLOGY AND LABORATORY MEDICINE, LHSC, WESTERN UNIVERSITY OBJECTIVES: 1. Understand the concepts of molecular generic methods and their application in diagnosis (PCR, Sanger, NGS) 2. Distinguish germline genetics from somatic genetics 3. Understand the role molecular and cytogenetic testing in cancer; Describe examples and clinical utility of genetic testing Recommended readings: "Robbins Basic Pathology" 11th Edition, 2023. Chapter 4 (pg. 79-230) - read relevant sections; basic genetics textbooks (e.g. Lewin’s Gene X). MOLECULAR GENETIC METHODS ① Polymerase chain reaction (PCR): make several copies of a specific DNA segment, which is exponentially amplified (chain reaction) Steps: 1. Denaturing of double strand DNA 2. Annealing of primers: (Primers that bind to the 3’ and 5’ ends of the normal gene sequence) 3. Elongation: Taq polymerase amplifies the DNA region Advantages: Fast; Use small amounts of DNA; Can multiplex; Detect a variety of mutations (deletions, insertions, point mutations) Limitations: Must know site of interest – need to know what is your mutation! understand these Size of allele that can be quantified ( therapy. In many improved disorders the carrier state can be identified to permit genetic counseling. By the testing of amniotic fluid before birth or appropriate screening at birth, these metabolic disorders can be detected. PATHOLOGY 3500/PATHOLOGY 9535 1 METABOLIC & NUTRITIONAL DISORDERS  in uterus W Since these disorders seldom occur, the in utero testing can usually assure parents that their child will be normal. Many of these metabolic disorders are characterized by autosomal recessive transmission and, in many cases, the chromosome on which the defective gene is located has been determined. Functional and pathological damage may be produced by loss of end product of a reaction due to enzyme deficiency, accumulation of substances prior to the metabolic block, or production of toxic metabolites. Indirect effects are also exerted on other metabolic pathways or functional elements. Some examples from this class of disorders will be discussed. These were selected because they are amenable to therapy or are representative of a class of enzyme deficiency diseases. #opena responsive Clinical Expression: The neurological complications present in many of these disorders range from specific focal abnormalities to mental retardation. The precise reason for the mental retardation is not clear. There is marked variation in the age of onset, rate of progression and organ and skeletal involvement among disorders and among variants of each disorder. This is due to factors such as the①different isoenzymes involved,② solubility of accumulated products for ⑬ excretion, and the specific biochemical reactions occurring in various organs. Phenylketonuria PKU is a disorder of amino acid metabolism in which the enzyme responsible for the conversion of phenylalanine to tyrosine is deficient (affects about 1 in 12,000 live births). This results in increased blood levels of phenylalanine, which impairs normal brain development, and increased glial cells in iNsproliferate urinary excretion of phenylpyruvic acid. Morphologically within the brain there is hypomyelination, - - when there is cell gliosis, and-microcephaly. There is no lysosomal storage in neurons. Is the are not that nevrons damage smaller than normal head "forms due to the excessive phenyl... myelinated Clinical features include severe mental retardation, seizures, and hyperactivity, as well as decreased pigmentation of the hair and skin (due to decreased melanin production from tyrosine). Because these consequences can be avoided by restriction of phenylalanine in the diet and supplementation with tyrosine, nearly all newborns are screened (e.g., Guthrie test-serum analysis). Many female PKU patients treated with diet early in life reach childbearing age and are clinically normal, but they may have markedly increased serum phenylalanine levels if no longer on a restricted diet. Children