Genetics Flipped lesson 8.PDF

()Genetics Flipped Lesson 8 – Clotting disorders and pharmacogenetics Coagulation cascade - Blood clotting - Plugs damaged blood vessels - Platelets + (migrate to site of energy) - Fibrin (Ia) - Fibrinogen (inactive form of fibrin) circulates in blood in high concentration - Activated to fibrin by t...

()Genetics Flipped Lesson 8 – Clotting disorders and pharmacogenetics Coagulation cascade - Blood clotting - Plugs damaged blood vessels - Platelets + (migrate to site of energy) - Fibrin (Ia) - Fibrinogen (inactive form of fibrin) circulates in blood in high concentration - Activated to fibrin by thrombin (IIa) - Complex cascade (chain reaction (activation of 1 factor catalyses the activation of the next)) involving 13 factors (mostly protease precursors) A blood vessel is made up of endothelial cells. If walls of blood vessels are damaged, cells no longer seal vessels from surrounding environment – to prevent blood pouring from vessel – platelets form a plug. “Plug” isn’t very solid so body needs a second mechanism to solidify it – fibrin strands act as a mesh to hold the platelet plug together. Fibrin units come together and make a strand – to ensure this only happens at sight of injury, fibrin doesn’t circulate in blood, fibrinogen does, which forms fibrin. Fibrinogen is converted to fibrin due to proteins that are released when blood vessel is damaged, which recruit thrombin, which is activated from an inactive form, prothrombin. XII →XI →IX + VIII →X + V → II → I (II = thrombin, I = fibrin, --- = intrinsic pathway. XII isn’t becoming XI, etc., they're catalysts for each other) III (TF (tissue factor)) → VII → X → II → I (-- = extrinsic pathway) Thrombin then activates V, VII, VIII, XI, XIII Fibrin molecules forms strands, connected by crosslinks. FXIII activates crosslinks. Extrinsic pathway is the “spark” – one activated by initial injury, intrinsic then does most of the coagulation Negative feedback to prevent clotting – thrombin creates plasmin from plasminogen – breaks up clots. Thrombin stimulates production of anti-thrombin – decrease amount of thrombin. - - 2 pathways o Extrinsic – Tissue Factor pathway -fast o Intrinsic – Contact Activation pathway - slower Common pathway after Factor Xa and Va activate Thrombin Importance of Thrombin - F VIIIa formed from F VIII by previously activated thrombin – deficiency = Haemophilia A - F IX activated by F XIa (also activated by thrombin) – deficiency = Haemophilia B - F Va formed from F V by previously activated thrombin – deficiency = Thrombophilia - Thrombin also activates F XI (near start of intrinsic pathway) and F XIII which crosslinks the fibrin strands together to form the clot at the end - (thrombin amplification loops) – positive and negative feedback control Case Presentation - 39 Year old man - Sudden onset of shortness of breath - Chest pain worse when he breathes in Haemophilia A - Gene F8 (factor VIII) - X q28 - 26 exons, > 186 kb DNA - Haemophilia A related to mutations in or near F8 - Mutations may result in o a null allele (no working product) o a hypomorph (product that works a bit) Haemophilia A : F8 Mutations - Gene F8: Types of Mutations 1. Large rearrangements: insertions, deletions 2. Small mutations (<50 bp, often SNPs) - 1800 reports of different F8 variants o mis-sense, non-sense, splice site variants - Classification of severity by clotting activity: o Mild – 5-40% o Moderate – 1-5% o Severe - <1% - Intron 22 of F8 has a sequence int22h-1 About 300 kb 5’ (upstream) of F8 gene is a complex structure that includes interspersed repeats in opposite orientation All 3 int22h sequences identical/very similar Haemophilia A – Large Rearrangements - int22h-2 and int22h-3 are flanked by imperfect palindromic sequences - Hybridisation & recombination can occur Haemophilia A - Inversions & deletios - Pairing between palindromic sequences (during male meiosis) can invert int22h-2 and int22h-3 - Recombination between copies of int22h result in 1. Inversion of F8 Exons 1 to 22 2. Deletion of F8 Exons 1 to 22 Inversion of F8 exons 1-22 Deletion of F8 exons 1-22 Haemophilia A Summary: - Chromosome architecture predisposes to a particular change (major inversion) which accounts for a high proportion of defective alleles and ~50% of severe cases - Approx 5% of severe cases related to deletions in F8 gene - Whole plethora or other less common mutations Role of Factor IX – Haemophilia B Haemophilia B /Factor IX - About 12% of haemophilia - Locus X q 27.1 - (Royal Families of Europe) - Queen Victoria was probably a de novo mutation (rare) - Wide range of mutations - About 10% of carrier females have <50% F9 and are at risk for abnormal bleeding Therapy for Haemophilia A/B - Haemorrhage prophylaxis o Pads, avoidance etc. - Local haemostasis (compression, sutures, etc.) - Clotting factors (pharmacogenetics) - Somatic gene therapy - Germ-line editing - CRISPR-Cas? Pharmacogenetics/genomics Pharmacogenetics: - The study of the impact of single genetic variants on drug metabolism - Predicting likely response and risk of adverse events based on mutation at a single locus Pharmacogenomics: - The study of drug metabolism in relation to the whole genome of an individual - Use of genomics to optimise selection of pharmaceutical agents for individual patients based on better prediction of likely response and risks of adverse effects Glucose 6 Phosphate Dehydrogenase Deficiency - Amongst most common genetic disorders (400 million) - Partial malaria resistance in hemi/homozygotes* - X-linked recessive – mostly affects males o Females affected via skewed XCI - Symptoms of haemolysis manifest when body is in oxidative stress caused by: o Infection o Medicines (including aspirin, sulfonamides, nitrofurantoin (UTI) and antimalarials) – pharmacogenetics o Foods e.g. fava beans – contain oxidants G6PDD G6PD Deficiency Symptoms - Haemolytic anaemia - Chronic haemolytic anemia - Acute Haemolytic Crises - Anemia and jaundice (hyperbilirubinemia) in the newborn - Kernicterus – irreversible neurological damage - Shortness of breath - Dark coloured urine - Prevention o Avoid triggers - Treatment o Bili-lights (newborns) - isomerises bilirubin o blood transfusion G6PD Deficiency Genetics - Gene map locus: Xq28 - Great heterogeneity of G6PDD alleles – more than 400 variants - Predominant alleles vary between ethnic groups o Different clinical severity associated with different alleles* - Prevalence of G6PD deficiency varies between ethnic groups o Common in areas with malaria o Africa, Mediterranean, Asia Things to Remember 1. The coagulation cascade is complex and mutations in the genes for many of the clotting factors can lead to heritable clotting disorders 2. Haemophilia A and B are classical examples of X-linked hereditary diseases 3. Haemophilia A is associated with F8 deficiency and Haemophilia B with F9 deficiency 4. Genome editing may hold promise for many diseases but there are many ethical issues, especially in germline editing 5. G6PD deficiency is a pharmacogenetic disorder that can lead to haemolytic anaemia, which can be prevented by avoiding triggers including certain drugs Question: Which of these statements is most suitable to describe the genetics of Haemophilia A? - It is an X-linked disorder that is associated with thrombin deficiency (no, mutations of F8 gene) - It arises in most cases due to deletions in the FVIII gene (no only 5%) - Severe haemophilia A is associated mainly with deletions involving intron 22 of the FVIII gene (no exon 1-22) - Palindromic interspersed repeats in and around the FVIII gene frequently pair in male meiosis leading to gene inversion or deletion