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Genetics & Disease By: Elham Sahibzada Overview • DNA Structure • Haemoglobin Structure • Mutations • Haemoglobinopathies • Cystic Fibrosis • Thalassaemia • Clinical Relevance to DNA Replication • Sickle Cell Anemia • Chromosomal Structure • Clinical Importance of Enzyme activity • Chromosomal Abnormalities • Molecular Techniques • Personalised Medicine • Polyploidy & Aneuploidy • Breast Cancer • Sex Chromosome Mutations • Monoclonal Antibodies • Translocation Mutations • Gene Therapy • Drug Repurposing • and Newbornrogrammes in Pregnancy • Pre-natal DiagnosticsDNAstructure • Glycosidic bonds within nucleotides • Phosphodiester bonds between nucleotides • Polarity; right-handed doublehelix; negatively charged (presenceof Phosphategroups); 5’to 3’ • Minor grooves + Major groovesDNAMutations CysticFibrosis Mutation in the geneCFTR ΔF508, is deletion of three nucleotides Mutationoutsidecodingregions 1. Inpromoter or enhancer* sequences ofa gene 2. Interminationsignals 3. Insplice donor andacceptor sites 4. Inribosome binding sites Phenylketonuria(PKU) β-thalassemia: A single base mutation(GT-to-AT) Splice-site mutations found inthe genomes change from 5'-GU to5'-AU inthe splice ofaffected patients donor site ofIntron12 = unrecognizable by the splicing enzymes ClinicalImportanceofDNAReplication Cisplatin Gemcitabine Mercaptopurine • Chemotherapy • Chemotherapy • Inhibit DNA agent agent synthesis - purine • Inhibit DNA • Inhibit DNA antimetabolite or synthesis via Elongation purine antagonist cross linking. • Treat: Crohn's Leads to disease, and apoptosis due to ulcerative colitis damage ChromosomalStructure • Indications ofKaryotyping; • Prenatal screening –chromosomal abnormalities • Birthdefects - malformation • Abnormal sexual development –Klinefelter Syndrome • Infertility • Leukaemia • set ofmetaphase/prometaphase chromosomee – • Karyogram –arranged inpairs; size, positionand centromere Normal Karyotype • detect changes inchromosome number and also more subtle structuralchanges • 46, XX • 46, XY ChromosomalAbnormalities • Polyploidy; > 2 sets ofchromosome (rare inhumans- lethal) • Tetraploidy; 4n • Endoploidy; 2n • Tetraloidy –polyspermy;miscarriage • Aneuploidy; abnormalnumber ofchromosomes • Trisomies (risk increases withmaternalage) • Caused:non-dysjunctionofchromosomes inmeiosis IinProphase I • ChromosomalMutations • RobertsonianTranslocation–monosomic/trisomic zygote (unbalanced gamates) • PhiladelphiaChromosome; fusiongene: BCR-ABL ChromosomalTrisomy's Trisomy 21; Downs Trisomy 18: Edwards Trisomy 13: Patau Syndrome Syndrome Syndrome • 47, XX + 21 • 47, XY + 18 • 47, XX + 13 • Most viable • 5-15 days life span • Mosaic Downs syndrome • SyndromeationDownsTrisomy 21; Downs Trisomy 18: Edwards Trisomy 13: Patau Syndrome Syndrome Syndrome SexChromosomeAneuploidies • Turner’sSyndrome • 45, X • Triple XSyndrome • 47, XXX • Klinefelter’sSyndrome • Able totolerate additionalsex chromosome than • 47, XXY extra autosomalchromosome dueto X • XYYChromosome chromosome inactivation. • 47, XYY • X chromosome inactivation(lyonization*) by transcriptionalsilencing occurs randomly for one of the twoX chromosomes infemale cells during development Barr body is aninactive X chromosome ina cellwith more thanone X chromosomeTurner’sSyndrome • A single X chromosome presents issues X chromosome are fully silenced.ivated • Pseudo Autosomal Regions(PARs)onthe X and Y chromosomes behave like autosomes and recombine during meiosis. • Turner syndrome patients lacking one X chromosome leads tohealthproblems. SexChromosomeMutations Klinefelter Triple X syndrome: 47, syndrome: XXY 47,XXXRobertsonianTranslocation • Breakage oftwo acrocentric chromosomes at or near their centromeres • chromosomeheir long armstocreate one • Results ina totalchromosome number reducedto 45 • Robertsoniantranslocationcarriers are typically asymptomatic • Nogainor loss ofcrucialgenetic material • Potentialfor unbalanced gametes production • Risk ofmonosomic ortrisomiczygotes Philadelphia Chromosome • Results from a reciprocal translocation between chromosomes 9 and 22 • Formation of fusion gene BCR-ABL, encoding oncogenic fusion protein BCR-ABL • Therapy primarily involves BCR-ABL tyrosine-kinase inhibitors (TKIs) • Commonly associated with chronic myeloid leukemia (CML) ScreeningProgrammeintheNHS Screening offeredduring pregnancy: 3 screening programmes that test forpre 1. Infectious diseases cancerouscell changes inthe body. 2. Inherited conditions 1. Inwomen: breast and cervical 2. Inbothmenand women: bowel 3. 11 physicalconditions (20 week scan) 4. Down’s syndrome,Edwards’syndrome and Patau’s syndrome Screening offeredfor the newborn 1. Newbornphysicalexamination 2. Newbornhearing screening 3. NewbornBlood spot screening ScreeninginNewborn Newborn Hearing Screening Newborn Physical Examination Newborn Blood Spot • Day 5 –Heelprick • The automatedotoacoustic Offered within72hrs of emission(AOAE) test. birth 1.Phenylketonuria(PKU) Examine: 2.Congenital • A smallsoft-tipped earpiece is • Generalhealth(colour, hypothyroidism(CHT) placed inbaby's ear and gentle tone) clicking sounds are played. • Eyes 3.Sickle celldisease(SCD) • Ifresults unclear offered a 2nd test. • Heart 4.Cystic fibrosis(CF) This may be repeat AOAE or another test called the automated • Hips 5.Homocystinuria(HCU) auditory brainstem response • Testicles (AABR) test. • Back • Hands/feet • Palate • Anus ScreeninginPregnancy Maternal Health screening Screening for Infectious Diseases • Ifclassified as highrisk mother offered Week 10 pregnancy 3 infectious further intervention: diseases • Gestationaldiabetes –OGTT 1. Hepatitis B • Preeclampsia –Low dose aspirin 2. HIV • MentalHealthdisorder –referralto 3. Syphilis specialist services Check immunity toinfectious diseases • Blood group and Rhesus Factor –AntiD due toprevious infections/immunisation(e.g. rubella) Enables early treatment toprevent verticaltransmission Screening for 11 Physical Conditions •20 Week scan •Detailed look at bones,heart,brain, spinalcord, face, kidneys and abdomen •Sonographer looking for 11 rare conditions Non-Invasive Prenatal Testing “Combined Test Quadruple Test Mid-pregnancy scan 10-14 weeks pregnancy 14-20w If presenttoo late for Combined Test Nuchal translucency Blood test (Alpha feta for T13and T18 protein, BHCG, Serum pregnancy-associated Unconjugated estriol and plasma protein A Inhibin A) Beta HCG • Down’s only • Less accurate Combined results of both tests calculaterisk for all 3 Trisomys Pre-natalDiagnostics Foetal DNA is isolated from: 1. Amniotic fluid cells 2. Chorion villus biopsy 3. Foetal DNA in mother's blood Amniocentesis 15–20 weeksofgestation Needle 90 degrees Ultrasound guidance Chorion villus biopsy 10–13 weeksgestation Foetalvillimust be separated from maternaltissue Haemoglobin&Myoglobin • Erythrocytes; live for about 120 days • Ironis bounded tothe protein by proximal histidine • Haldane effect • Myoglobin; Hyperbolic curve • Haemoglobin; Sigmoidcurve due toCooperative Binding ofoxygen Methaemoglobinaemia • BOHREFFECT - The Bohr effect ensures the delivery ofO2 is coupled todemand. • Carbonmonoxide (CO)is a poison because it combines withferromyoglobin and ferrohaemoglobinand blocks oxygen transport. • FatalwhenCOHb is >50% • turns skina bright cherry colour that 2-3 Biphosphoglycerate decreases haemoglobin’s oxygen persists after death affinity Shifts the haemoglobinoxygencurve to the right Chronic hypoxemia Haemoglobinopathies-Thalassaemia • Autosomal RecessiveGenetic mutation/deletioninthe Alpha or Beta Globingenes for Haemoglobin. • tropicaland subtropicalregions • Heterozygotes inthose regions have protectionfrom malaria hence highand maintained carrier frequency β0 :NoBeta globinchains produced by this gene β+ :Some Beta globinchains produced by this gene ThalassaemiaManagement Management: Complications: 1. Long term folic acid • IronOverload(eg. From frequent blood transfusions) 2. Regular transfusions if needed to keep the Hb • Damage toheart/liver/endocrine organs. above 100g/L • Infection(Especially post splenectomy) 3. Consider Splenectomy • Bone Deformities (Thalassemia cancause physical 4. Management of iron overload bone marrow expansionà abnormalbone structure;especially inface and skull) 5. Consider bone marrow transplantation • Splenomegaly (May require splenectomy) 6. Prenatal diagnosis • Slowed growth& delayed puberty • Congestive heart failure 7. Care by specialist MDT SickleCellAnaemia • Haemoglobinvariant (HbS) • Glutamic acidto valine at position6 of β globinchain • Autosomal RecessiveInheritance - Prevalent inAfro-Carribean populations. • Essentially asymptomatic • 2x αchains, 1x βSchain,1x β chain, 4x Sickle Cell Crisis: heme Vaso-occlusive crisis Acute chest syndrome Splenic sequestration Haemolysis Bone marrow aplasia SickleCellManagement Risk of iron overload due to ineffective erythropoiesis. Regular transfusions in such patients Acute Management Long term Management contribute to excessive iron absorption. • Painrelief • PenicillinVprophylaxis • IVFluid • Vaccination The body lacks a natural mechanism to eliminate excess iron. • Oxygen • Folic acid • Antibiotics • Hydroxycarbamide Iron overload can lead to toxicity • Exchange transfusion • Preventative transfusions Treatment options include oral and • Stem celltransplantation subcutaneous iron chelators such as deferoxamine, deferiprone, and deferasirox, Long-term compliance is necessary ClinicalImportanceofEnzymeActivity Drug targets Angiotensinconverting enzyme inhibitors (e.g. Enalapril) Biomarkers Liver functiontests: alkaline phosphatase (ALP) and γ-Glutamyltransferase (GGT) Enzyme deficiencies Disruptiontometabolic pathways leading to significant clinicalconsequences Phenylketonuria (PKU) –low levels ofthe enzyme phenylalanine hydroxylase and the inability to metabolize phenylalanineMolecularT echniques-AnalysisofDNA/RNA • PCR • DNA/RNA electrophoresis • Restriction analysis • DNA sequencing • Karyotyping • FISH • DNA HybridisationMolecularT echniques-AnalysisofDNA/RNA Polymerase Chain Reaction 1. AmplificationandanalysisofDNA from a single cell, hair follicle, sperm for investigations inforensic medicine 2. Diagnosisandgenetic analysis ofinherited diseases suchas β-thalassaemia,sickle celldisease,haemophilia, Tay-Sachs disease 3. Diagnosis and analysis ofneoplastic disorders 4. Prenatal andpre-implantationdiagnosis 5. Multiplexpolymerase chainreaction is a technique utilized for the amplificationofmultiple targets MolecularT echniques-AnalysisofDNA/RNA Restriction Fragment Length Polymorphism & Gel Electrophoresis • Separate charged molecules like DNA, RNA and proteins according totheir size • RFLP (RestrictionFragment Length Polymorphism) • for Sickle cell anaemia - single base mutationremoves a restrictionsite within a gene • for Junctional EpidermolysisBullosa restrictionsite withina genecancreate aMolecularT echniques-AnalysisofDNA/RNA DNA hybridisation - FISH (Fluorescence in situ hybridisation) 1. Toinvestigate genesinsitu: deletion/duplicationor amplification 2. Toinvestigate chromosome ; deletions/duplications or translocations 3. Toinvestigate chromosome number: e.g. trisomy 21MolecularT echniques-AnalysisofProteins • Protein gel electrophoresis • Serum proteins electrophoresis • Protein electrophoresis used to detect allozyme variation MolecularT echniques-AnalysisofProteins Monoclonal Antibodies Antibodies are used indiagnostic tests(eg. autoimmune disorders, infectious and metabolic diseases) therapy totreat MS,rheumatoid arthritis,cancer) monoclonalantibody ELISA (Enzyme-Linked Immunosorbent Assay) • Detectionof platelet antibodiesinserum used toidentify patients suffering from idiopathic thrombocytopenic purpura and systemic lupus erythematosus • Pregnancy testfor the detectionofhormone humanchorionic gonadotrophin(hCG) inurineDisease Analyte Technique(s) Sickle celldisease Haemoglobins,Hb Mass Spec or Electrophoresis or HPLC Cystic fibrosis Immunoreactive trypsinogenIRT ELISA Hypothyroidism thyroid stimulating hormone TSH ELISA Phenylketonuria (PKU) Phe (Tyr) Mass Spec, (enzyme assay) Homocystinuria Met (homocysteine) Mass Spec Maple syrup urine disease (MSUD) Leu,Ile, alloisoleucine Mass Spec PersonalisedMedicine 1. PredictionandPrevention ofdisease - FamilialHypercholesterolemia (FH) 2. More Precise diagnoses - MultiparametricMRI 3. Personalisedand targeted interventions - Warfarin& CYP2C9 genotype 4. Participatory role for patients - Abacavir specific genomic test for allergic reactions CancerBiomarkers Biological moleculefound inblood, other body fluids, or tissues that is a signofa normal or abnormal process, or ofa condition or disease, suchas cancer BreastCancer • Tamoxifen; metabolized via CYP2D6 into endoxifen, its primary active metabolite that blocks ER • Trastuzumab(Herceptin) is a humanised monoclonalantibody against HER2 receptor • Oncotype DXtest for early stage, ER+, HER2- breast cancers • Analyzes expressionof16 cancer-related genes • Provides informationoncancer behavior and treatment response • Serves as bothprognostic and predictive test • Predicts likelihood ofbenefit from chemotherapy or radiationtherapy Personalised Med. - Monoclonal Antibodies Bevacizumab Immune Checkpoint Cetuximab Trastuzumab Inhibitors (Herceptin) (Avastin) • Chimaeric monoclonal • Humanisedantibody •T cell targeted antibody against EGFR • Humanised toVEGF(Vascular monoclonal EndothelialGrowth immunomodulators • Metastatic colorectal antibody blocking the immune cancer, metastatic non- • against HER2 Factor) •checkpoints CTLA-4 smallcelllung cancer and receptor • Treatment of andPD1 or PDL1 head and neck cancer treatment ofHER2 colorectalcancer, positive breast nonsmallcelllung cancer carcinoma, renal cancer, glioblastoma and ovariancancer Therapeutic Cancer Vaccines Preventative Cancer Vaccines: HPVvaccine: induce productionofneutralising antibodies that block the humanpapillomavirus virions Ex-vivoGeneTherapy CART-cell Therapy • Used for treatment ofB-cellacute lymphoblastic leukaemia (B-ALL) in childrenand young adults • diffuse large B-celllymphoma (DLBCL) inadults STRIMVELIS • treatment ofsevere combined immunodeficiency due toadenosine deaminase deficiency (ADA-SCID), a rare autosomalrecessive disorder ZYNTEGLO- treatment ofBeta-thalassaemia Autologousexvivo therapy for Recessive Dystrophic • Epidermolysis Bullosa (COL7A1 encoding collagenVI) - - A strategy combining gene therapy and a tissue-engineered skinsubstitute (TES) In-vivoGenetherapy Splicing Therapy for Duchenne LUXTURNA (voretigene ZOLGENSMA Muscular Dystrophy neparvovec) (onasemnogene abeparvovec) • affect the largest gene inthe humangenome, • Retinal dystrophy is a rare DMD (2.4 Mb)that encodesdystrophin, autosomalrecessive • Used for treatment of • DeletionsofDMD exons 48–50 lead to disorder caused by mutation spinal muscular productionofa spliced mRNA ingene encoding for RPE65 atrophy (SMA) (retinalpigment epithelium- • mutationinthe SMN1 associated protein) resulting gene inblindness • LUXTURNA uses theadeno- associatedviral vector serotype 2 (AAV2) tocarry a functionalcopy ofthe RPE65 gene intothe retinalpigment epithelial(RPE) DrugRepurposing Aspirin Thalidomide Sildenafil • Initially analgesic • Initially:sedative andto • Antihypertensive drug and as alleviate morning sicknessin treatment for angina • Now:antiplatelet pregnant women. aggregationdrug • Anunexpected side effect • soonbe repositioned • Repurposed in1998 as an emerged during clinicaltrials againinoncology orphandrug forleprosy inthe form of penile complications erectionsdue to • Inhibits synthesis of vasodilatation proinflammatory TNF-α • treatment ofthe idiopathic form of pulmonary arterial • Second repositioning in2006 as hypertension first-line treatment formultiple myelomaFeather, A. et al. (2021) “Haematology,” in Kumar & Clark's Clinical Medicine. Amsterdam: Elsevier Boron, W.F. and Boulpaep, E.L. (2017) Medical physiology. Philadelphia, PA: Elsevier. Molecular Biology of the Gene. James Watson et al. (7th Ed., 2013; ISBN 978- 0321762436) •Essential Cell Biology. Bruce Alberts et al. (4th Ed, 2013; ISBN 978-0815344551) Marks’ Essentials of Medical Biochemistry. Michael Lieberman. (2dn Ed, 2014; ISBN 978- •1451190069)