md210 genetics mcq2.pdf

Genetics Flipped Lesson 6 – Molecular Genetics and Sequencing Molecular Genetics Definition - Refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA Molecular genetics for diagnosis - 2 main approaches: o Nucleic acid hybridisation – based on complementa...

Genetics Flipped Lesson 6 – Molecular Genetics and Sequencing Molecular Genetics Definition - Refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA Molecular genetics for diagnosis - 2 main approaches: o Nucleic acid hybridisation – based on complementarity o Nucleic acid amplification – making many DNA copies - 4 categories of test: o Mutation scanning – is there any mutation present? o Mutation screening – is a known mutation present? o Genomic quantification – is there extra/missing DNA? o Sequencing – what is the exact DNA/RNA sequence? Hybridisation - Sense (coding) and antisense (template) strands are hybridised to each other in a dsDNA molecule - When heated to 95°C the strands denature (break apart) - Complimentary sequences can then hybridise to each strand Amplification (PCR) - Primer sequences can hybridise (anneal) to each strand at 5560°C - In the presence of dNTPs and DNA polymerase the complimentary DNA strand is extended in each direction (at 72°C) - The resulting 2 amplicons have the same sequence (copies) - Each cycle doubles the number of amplicons - amplification Exponential amplification of the target DNA sequence Mutation Scanning Methods - Is there any mutation present? - Examines a gene for all possible mutations (including unknown) - Based on analysis of physical or chemical characteristics of PCR product (hybridisation and amplification) - Abnormalities must be confirmed by sequencing - Methods include: o SSCP (single-strand conformation polymorphism) o DGGE (denaturing gradient gel electrophoresis) SSCP and DGGE Mutation Screening Methods - Is a known mutation present? - PCR based screening for known specific mutations - Indicated in diseases with few discrete, but frequent mutations - Need to know sequence you are looking for to design specific primers - Can check presence/absence and size of specific sequence of 50bp to 5kb (RFLP) - Methods include: o PCR o OLA o Southern Blotting/RFLP Polymerase Chain Reaction - Detects specific known mutations - Can check presence/absence and size of PCR product Allele-specific PCR and the oligonucleotide ligation assay Southern Blotting/RFLP Detects large scale changes in DNA fragments e.g. long repeat extensions in triplet repeat disorders Genomic quantification methods - Is there extra/missing DNA? - Can look at whole genome on one microarray - Detect duplications, heterozygous deletions, copy number variability, unbalanced structural abnormality at resolution of 30kb - Indicated in cases of multiple malformations or unknown dysmorphism - Limitations of arrays – can’t detect balanced rearrangements (inversions and translocations), small deletions or duplications, point mutations, expanded trinucleotide repeats. Not suitable for most monogenic disorders. - Methods: o DNA Microarrays o SNP arrays (SNP chips) DNA Microarray Analysis (CGH) Di-deoxy (Sanger) Sequencing Next Generation Sequencing (NGS) - Massively parallel sequencing technologies - Ultra-high throughput - High accuracy and flexibility - Dramatically cheaper than Sanger - Large range of “omics” applications o Metagenomics o Proteomics o Epigenomics Next Generation Sequencing A Word of Caution - Molecular genetic testing can provide a lot of information, both wanted and potentially unwanted - Need to consider how this could affect the patient, especially with regards to predictive testing - Sensitivity (true positive rate) and specificity (true negative rate) are important factors to consider when choosing a test. Things to Remember 1. Molecular Genetics refers to all diagnostic methods that examine changes of genetic information in extracted DNA or RNA 2. DNA array analysis relies on the principle of hybridisation and is useful for detecting any small unbalanced structural abnormality and numerical abnormalities 3. Next Generation Sequencing technologies hold promise for the diagnosis, prognosis and therapy of genetic diseases 4. Genetic testing can provide a lot of information – need to consider what is necessary information for the patient when choosing the test Question: What is the best type of molecular genetic test to do for a foetus with suspected Sickle Cell Disease? - A mutation screening test such as allele specific PCR or OLA - Sequencing of the β globin gene - A mutation scanning test such as SSCP - A genomic quantification test such as a DNA array MD210 Genetics Flipped Lesson 7 – Epigenetics Epigenetics - Heritable/Enduring change in gene expression not related to variation in nucleotide sequence (A, C, T & G of nucleotide sequence don’t change but a chemical modification occurs that changes the expression of the gene sequences. Related to chromatin confirmation.) - May be enduring in the sense of: o the life of a long lived cell o retained from mother to daughter cell (X-inactivation) o retained from parent to offspring (imprinting) - Epigenetics relate to DNA factors other than nucleotide sequence that impact on gene expression Epigenetics biochemical basis (Readability of genes based on chromatin structure. Condensation of chromatin and packaging density of DNA are regulated by chemical modifications of histones. Chemical modifications of histones influenced by DNA methylation. DNA methylation occurs in regions of DNA known as CPG islands – nucleotide sequences rich in cytosines and guanines, usually located in the promotor regions upstream of genes.) DNA Methylation - Gene “switched on” o Active (open) chromatin o Unmethylated cytokines (white circles) o Acetylated histones (Transcription machinery can get in, bind to the promotor, and transcribe the gene) - Gene “Switched off” o Silent (condensed) chromatin o Methylated cytosines (red circles) o Deacetylated histones (tight wrapping of nucleosome). Biallelic/Monoallelic Expression - Humans Are Diploid - 23 Pairs of Homologous Chromosomes (1 homolog inherited from each parent) - 2 Copies (often different alleles) of each gene - Biallelic expression – both alleles expressed - Monoallelic expression – only one of the two alleles is expressed Imprinting - Involves Monoallelic Expression - The expression of genes in a parent-of-origin-specific manner - Only the allele inherited from a specific parent (either the maternal or paternal allele) is expressed for imprinted genes - <1% of human genes are imprinted (rare) Imprinting - Imprinting is related to Methylation of Cytosines (in promoter) & histones - Modification of DNA but not nucleotide sequence change (epigenetic) - Enduring, but not permanent change (can last from cell to cell, mother to daughter, but not permanent) Genomic Imprinting (E.g. Chr 1. In the body cells will have 1 copy of each from both parents. In the germ cells the imprint pattern will be deleted. For every Ova, all chromosomes rewritten with a sex specific imprint – maternal imprint rewritten onto Ova. All paternal imprint patterns deleted in primordial germ cells – all sperm rewritten with paternal imprint. In the zygote have 2 homologs, 1 with maternal imprint and 1 with paternal imprint.) Imprinting example: IGF2 (How imprinting works to control the monoallelic expression of genes) - Insulin Like Growth Factor 2 (IGF2) (Gene involved in growth cycle) - Only the Paternal Allele is Normally Expressed for this gene - The Maternal Allele is “Imprinted” (shut down) (not transcribed and translated into protein) (In image – Homolog of Chr 11 from father, written with paternal methylation pattern on IGF2 – will be active – euchromatin – it will transcribe into mRNA and the mRNA will translated into the IGF2 protein. Maternal homolog also has IGF2 but its written with a maternal methylation pattern – its imprinted – its going to be shut down, wont be transcribed into mRNA and wont be translated into a protein. Stops too much IGF2 being produced and stops the cells from growing out of control) Clinical example - The Drapers Imprinting & Sally - Sally has an allele of Insulin Like Growth Factor 2 (IGF2) from Don – which works (paternal allele) - She has an allele of Insulin Like Growth Factor 2 from Betty that is switched off (imprinted (because was written with the maternal pattern)) Question - If Sally has a child, which allele of the IGF2 gene will work? - In any oocytes that Sally (the mother) makes, whichever version of the allele is present will be switched off (her ova will be rewritten with the maternal methylation pattern) - Imprints are erased in the Germline and re-established in gametes In the sperm all imprints are erased and rewritten with the paternal pattern, even the alleles that came from mum In the ova all imprints are erased and rewritten with the maternal pattern, even the alleles that came from dad Diseases Related to Imprinting - For imprinted genes, the inheritance of maternal and paternal alleles is required for normal development (need both a maternally imprinted homolog and a paternally imprinted homolog – some genes expressed from both) - Imprinted genes are more vulnerable to mutation – haploid expression (only have 1 copy of gene with the correct methylation pattern). (if there is a mutation in the paternal homolog – it’s the paternal homolog that’s usually expressed – wont have any functional gene – maternal homolog will still be shut down and cant be switched back on) - Dominant mutations (dominant inheritance patterns) Angelman Syndromes - Small - Profound intellectual disability - (unable to speak and with particular behaviour pattern (characterised with a happy, friendly demeanour, inappropriate laughter)) Prader-Willi Syndrome - Floppy babies (hypotonia) - Intellectual/cognitive disability (not as profound as Angelman) - Uncontrollable appetite (often leads to obesity) - Hypogonadism in males - (Skin-picking behaviour that causes scabs and scars) - Study in the UK in 2000 estimated birth incidence to be in the region of 1:22,000, previous estimates have estimated a birth incidence between 1:10,000 and 1:25,000. - Uncertainty is due to the fact that PWS may still go undiagnosed - Note: Ascertainment & the reported incidence of illness (often vary significantly from what they actually are) Angelman, Prader Willi and Imprinting - Both associated with a de novo deletion on long arm of Chr 15 (15q11-q13) - Both usually for identical sets of genes but…(depends on which homolog it involves) - Deletion from paternal Chr 15 = Prader Willi - Deletion from maternal Chr 15 = Angelman How does that work? Another way it can happen – Uniparental Disomy (UPD) - UPD occurs when 2 copies of a Chr come from the same parent - Therefore both will have either maternal or paternal pattern of methylation (missing the expression of either 1 set of genes involved in Prader Willi syndrome or the other set involved in Angelman) Mechanisms of UPD (Trisomy rescue – zygote that has 3 copies of the same chromosome, the paternal copy is removed to make it a normal disomy zygote instead of the extra maternal copy being removed. Gamete complementation – one germ cell missing a copy of a chromosome, the other can compliment it by having a duplicate of a copy. Monosomy rescue – if there’s one chromosome missing because 1 germ cell didn’t have a chromosome, the other chromosome can duplicate itself so that there are two homologs from the same parent. Monosomy rescue – if 1 of the copies are faulty it can be excluded and can get duplication through the functional copy but it will have the same methylation pattern.) X-Inactivation (another epigenetic phenomenon) - The random transcriptional silencing of all but one X chromosome in females (X chromosome is very big, contains a lot of genes compared to Y chromosome – gene dosing. 2 copies of X chromosome in female that are both active there will be a lot of genes expressed to a much higher extent in females than males. X-inactivation means 1 X chromosome in each cell is transcriptionally silent) - Heritable from mother cell to all daughter cells in an individual (once X inactivation happens in one cell, all of the daughter cells of that cell have the same copy of that X inactivated.) - Not heritable from parent to child Human Embryonic Development X inactivation happens around time of late blastocyst formation when differentiation of layers starts X Inactivation - Early in Embryonic Development (totipotent cells) both X chromosomes are active - With differentiation to pluripotent cells random X inactivation (late blastocyst stage) - Inactivated X condensed on periphery of nucleus (Barr body (aka sex chromatin)) X Chromosome Inactivation - Random inactivation of paternal or maternal X - The inactivation is retained through all subsequent mitosis – all daughter cells - But not retained across generations - XIST gene in the X inactivation centre (Xic) produces Xist RNA transcript that covers the X chromosome to be inactivated (Xi). - Subsequent DNA methylation (epigenetic) then makes this silencing permanent in all daughter cells. Skewed X-inactivation - Ratio of inactivated maternal X to paternal X should be equal (random), but is uneven (skewed) in 10-15% of women - Preferential inactivation of the “abnormal” X in heterozygotes is the reason why there is such variability in the expression of X-linked disorders. - If the diseased X-linked allele does not cause selection, the extent of the primary stochastic skewing can influence the severity of the disease. - Skewed X-inactivation common in cases of severe X mutation or structural anomaly Rett Syndrome - X-linked dominant disorder - Usually caused by mutations in MECP2 gene (X Chr) - Affects young girls from 6-18 mo o Brain/cognitive function impaired o Hand coordination lost (hand-wringing) o Seizures and breathing problems - Substantial phenotypic variability - May be influenced by Skewed X-inactivation - Skewing >80% is rare o Asymptomatic/less severe carriers Duchenne Muscular Dystrophy - X-linked DMD – dystrophin (Xp21.2) - Muscle fibre weakness that presents in childhood - Difficulty walking and rising (Gower manoeuvre) - Wheelchair by teens - Respiratory weakness - Mild cognitive impairment - Cardiomyopathy (ventricular remodelling) - Heterozygous female carriers: - Mild cardiomyopathy, increased CK and muscle weakness but don’t present with DMD Those who do may have skewed X-inactivation Things to Remember 1. Epigenetics relate to DNA factors other than nucleotide sequence that impact on gene expression (primarily methylation of CpG dinucleotides) 2. Imprinting involves monoallelic expression of genes in a parent-of-origin specific manner. Imprinted genes are shut down. All imprints are erased in the germ line and renewed in the gametes. 3. For imprinted genes, the inheritance of maternal and paternal alleles is required for normal development. Imprinted genes are more vulnerable to mutation – haploid expression (dominant mutations) 4. Angelman and Prader Willi are 2 syndromes related to imprinting of Chr 15 5. X-inactivation is the random transcriptional silencing of all but one X chromosome in females 6. Skewed X-inactivation occurs in 10-15% of women. Preferential inactivation of the “abnormal” X in heterozygotes is the reason why there is such variability in the expression of X-linked disorders. Question With regards to X chromosome inactivation, which of the following is NOT true A. The inactivated X chromosome forms a Barr body B. Either the maternal or paternal X chromosome is inactivated at random in each pluripotent cell C. The XIST gene is transcribed from the X-inactivation centre on the X-chromosome that will be active D. Subsequent DNA methylation is what makes X-inactivation permanent in all daughter cells MD210 Genetics lecture 7: Epigenetics Mosaics - A mosaic is an individual who arose from a single fertilised egg, but who has 2 or more populations of cells each with different genotypes (Females are mosaics – 2X chromosomes - x-inactivation – some cells express X from mother, some express X from father) Clinical case – Gwen Dooley - 10 year old girl - Parents are quite tall - Born with puffy feet and hands - Scoliosis - Development normal, but small stature - Always smallest child in class Family History None of Gwen’s sibs or extended family show signs - Gwen’s lymphedema was apparent from birth, but small stature was only noticed from age of 5 Traits/Symptoms - Coarctation of aorta or bicuspid aortic valve - Webbed neck and lymphedema (sometimes) - Kidney problems – horseshoe kidney - Amenorrhea - Non-functional ovaries (streak gonads) – infertile - Non verbal learning disabilities and behavioural problems – variable Turner Syndrome (aka monosomy X) – causes - Turner syndrome occurs due to anaphase lag – where one sex chromosome moves too slowly to the pole of the daughter cell during division - It can happen: o During gametogenesis - classical monosomy X – 45,X o During early mitotic division - Turner Mosaicism – 45X/46XX or 45X/46XY (some are normal and some are missing a sex chromosome) o Neither is inherited Treatment options and priorities? - Depends on symptoms and level of mosaicism – screening and management of comorbidities o Heart problems o Scoliosis o Ear infections o High blood pressure o Thyroid o Bone loss - Neuropsychological and psychosocial issues - Hormone treatment - Infertility treatments Key investigations - Karyotype – translocation - Cardiac function tests - Cardiac imaging o CT, MRI, MRA - Y chromosome PCR Genetic counselling issues? - Impact of infertility o IVF and ERT - Not hereditary – reassurance for parents/Gwen - What if she is Y positive? (perform Y chromosome test and if necessary removal of streak gonads so they don't become malignant) - Transition of young people with TS to adult care o Advocacy and peer support o Ongoing multidisciplinary care Why is 45,X a problem? - X-inactivation is incomplete o Gene dosing - Up to 25% of X chromosome genes partially or totally escape (including PAR genes, e.g., SHOX) o Genes outside PARs may contribute to female phenotype - Escape genes expressed in higher levels in normal women versus Turner women Scenario - Mr and Mrs X - IVF Baby Born June 2014 - Healthy Male - Blood Group AB - Mother Group A (AA or AO) - Father Group A (AA or AO) - Cause? o They Took the Wrong Baby Home ? o Has Mrs X had another relationship ? But baby born by IVF o The Clinic Used the Wrong Sperm? Except they believe that this is Impossible o Paternity Test: ▪ Results Are Not Consistent With Mr X being the father ▪ More Detailed DNA Analysis Suggest Mr X is the Uncle - Mr X is a chimaera - Tetragametic Chimerism 2 Oocytes + 2 Sperm – 2 zygotes that merge into one organism - Mr. X has 2 genomes - Some cells carry the Allele A – make his red blood cells - Some cells carry the Allele B – but were not included in Mr X's haematopoietic tissue - Some of his brother’s cells were included in his gonads – carried his brother's genome Chimaerism vs Mosaicism - Chimaera = an individual with 2 or more populations of cells with different genotypes who arose by fusion of more than one fertilised zygote during embryogenesis (very rare) - Mosaic = an individual with 2 or more populations of cells with different genotypes who arose from a single fertilised egg - Usually result of a somatic change during early replication - Can be result of Germline mosaicism – occur early in germ cell development, resulting in a significant no. of gametes that carry the mutation and thus can affect >1 child Imprinting – Example – Robert Draper - Robert has an allele of Insulin Like Growth Factor 2 (IGF2) from Don (his father) – which works - He has an allele of Insulin Like Growth Factor 2 from Betty (his mother) that is switched off (imprinted) - If Robert has a child, which allele of the IGF2 gene will work? - It could be either- whichever allele, from either of his parents, that is in his sperm gets switched on – all imprinting is erased and rewritten with Roberts pattern in gonad's o 1. Roberts’ body cells ▪ Paternal IGF2 expressed (paternal methylation pattern) ▪ Maternal IGF2 imprinted (maternal methylation pattern) o Roberts’ primordial germ cells o Paternal and maternal methylation patterns deleted from both chromosomes o Roberts’ gametes ▪ Paternal imprint rewritten on both chromosomes in all sperm Why is this important? - IGF2 functions in regulating growth during gestation - If both alleles should begin to be expressed in a cell, that cell may develop into a cancer - E.g. Wilms’ tumour is an embryonic kidney cancer associated with loss of imprinting (LOI) of maternal IGF2 Angelman, Prader Willi and Imprinting - Both associated with a de novo deletion on long arm of Chr 15 (15q11-q13) - Both usually deletions for identical sets of genes but… - Deletion from paternal Chr 15 = Prader Willi - Deletion from maternal Chr 15 = Angelman Question: - What would a child with paternal uniparental disomy for Ch 15 have ? - Child is missing maternal Chr15 – Angelman's syndrome Prader-Willi & Angelman & Imprinting Errors - An affected individual may have BOTH maternal and paternal chromosomes - BUT – In Prader Willi Syndrome o the paternal chromosome may have maternal pattern of methylation (effectively no paternal chromosome) - In Angelman o The maternal chromosome may have the paternal pattern or methylation - Epigenetic (imprinting) error - Mutations in the imprinting control centre Things to remember - A mosaic is an individual who arose from a single fertilized egg, but who has 2 or more populations of cells each with different genotypes - Mosaicism is usually somatic, but can occur in the germline and then there is an increased risk for siblings and offspring - Chimaera = an individual with 2 or more populations of cells with different genotypes who arose by fusion of more than one fertilised zygote during embryogenesis - X-inactivation is incomplete - up to 25% of X chromosome genes partially or totally escape (including PAR genes), which has implications for gene dosing - Diseases related to imprinting can occur due to UPD or a mutation in the imprinting control centre, as well as deletion/mutation of the genes themselves on the relevant Chr ()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 MD210 Genetics Lecture 8 – Clotting disorders and Pharmacogenetics Case Presentation - Ben, second child born to parents Richard and Jenny - Bumped head created a large cephalohematoma - Jenny's two maternal uncles have mobility problems due to haemarthrosis in their knee and ankle joints - Jenny's mother and sister are fine - Ben's sister is also fine X-linked recessive – haemophilia – but which type? Ben's coagulation test results: - Bleeding time = 5 min - Prothrombin time (PT/INR) = 1.0 (measure of extrinsic pathway – measured in a ration of patient to “normal”) - Partial Thromboplastin Time (PTT) = 50 sec (slightly raised) - F VIII activity = 0.5% - F IX activity = 90% - What has he got? Haemophilia A – characterised by F8 deficiency Explainer – coagulation test - Partial thromboplastin time (PTT) (intrinsic/common pathway) o Time it takes plasma to clot when activator (e.g. silica) is added o Normal = 20-35 sec - Prothrombin Time (PT)/international normalised ratio (INR) (extrinsic) o Time it takes plasma to clot after addition of tissue factor o The INR is a standardized measure of this time, which compares patient’s PT to the PT of a normal control sample o Normal = 0.8-1.2 Haemophilia A - Genotype & Phenotype - For each male proband there is a mean of 5 females requesting carrier status determination - Where do we start? - Phenotypic tests may help - measure F8 activity, PTT (intrinsic/common), PT/INR (extrinsic) - “Carrier” females may have disordered coagulation tests or even mild clinical bleeding in tendency (skewed X inactivation) – approx. 30% - Rarely females have severe haemophilia due to skewed X-chromosome inactivation or a disorder of inactivation of X chromosome Haemophilia A & Carrier Women - F8 allele is expressed from whichever X chromosome is active in a given liver cell - One functional F8 allele in most women = 50% activity - Phenotype = clotting - About 10% of carrier females have <40% activity, at risk for abnormal bleeding - 2 non-functional F8 alleles = no functional gene - Phenotype = bleeding Haemophilia A - Assessing Carrier Status: - Genetic methods: 1. Direct sequencing of F8 (a big job but rapidly getting less so) 2. Linkage Markers within F8 o Dinucleotide repeat in intron 13 and 22 (microsatellites) o Known SNP’s in introns 18 and 22 o Need to compare to affected family members Problem A 10 week pregnant female who is a carrier of a Haemophilia A mutation in F8 comes to your clinic to request prenatal testing of her baby. What should you do? - Amniocentesis – risky - Chronic villous sampling – too intrusive - Determine gender of baby – males more likely to be affected Prenatal testing - Initially – test maternal blood for Y Chromosome DNA at 10 weeks - If Y is not detected then chronic villus sampling (1% risk of loss of pregnancy) not required - If Y detected the person may request chronic villus sampling (advantage: prepare for birth of baby – use precautions) - What else is testing for Y Chr used for? Turner Syndrome Case Presentation - James 22 - As a neonate suffered a cephalohaematoma - F IX activity at birth 0.5% (FIX associated with haemophilia B – but doesn’t usually get better) - Increased to 3% at 1 year - 19% at 12 years - No bleeding - 32% at 18 years - Mutational analysis revealed (c.-35G>A) (translation – typically promotor region of gene) - What has he got? Factor IX Leyden - A rare Haemophilia B sub-type (~3%) - Androgen therapy or puberty rises F IX activity from <1% to 30% -60% (fine for a normal clotting phenotype) - Associated mutations (>20) from -40 to + 20 of F IX - This promoter sequence resembles an androgen response element Case Presentation - Carla 23 - Suffers labrum tear during match - Bleeding is profuse during surgery to repair prompting investigation - Coagulation tests: o FVIII – 60% o FIX – 95% o PTT – 40 secs o PT/INR – 1.0 (INR = international normalised ratio) o Bleeding time 20 min - What has she got? Von Willebrand disease (FVIII, associated with adhesion of platelets – bleeding time) Von Willebrand Factor - In plasma, VWF and FVIII circulate as noncovalent complex that regulates platelet aggregation and clot formation - VWF is required for platelet adhesion to subendothelium, and for normal FVIII survival in circulation - VWF protein is not an enzyme - Binds to and inactivates FVIII – increasing serum half-life from 1-2h to 8-12h - Thrombin releases FVIII from VWF and activates platelet receptors so VWF and activates platelet receptors so VWF can bind, facilitating platelet adhesion Von Willebrand Disease - Most frequent congenital clotting disorder (1% prevalence) - VWD Chr 12p13.3 - large gene – 52 exons 178 Kbp - Many alleles - heterogeneity of subtypes - Quantitative or qualitative defects of VWF - Inheritance can be dominant or recessive - Broad clinical phenotype spectrum: - Asymptomatic/mild (common) to severe haemorrhaging (rare) - Treatment – Desmopressin or “factor” - Often reduced FVIII levels: (but not to same extent as Haemophilia A) - Metabolised faster without VWF - Also - faulty thrombocytic adhesion > increased bleeding time Thromboembolism - Formation of thrombus in deep veins (typically of leg) - Pain & Swelling of the leg - Thrombus may become detached from the vein (embolus) and travel to the pulmonary artery - Embolus wedged in pulmonary artery – obstructs blood supply to a segment of lung – pulmonary - Obstructs main pulmonary artery – sudden death (too much clotting instead of not enough) Factor V Leiden - Dutch city of Leiden, late 1980s early 1990s - Investigation of venous thromboembolism in patients without identifiable risk factors - Identified a group of people with poor response to activated protein C (APC) in a coagulation assay Factor V Leiden Thrombophilia - Found a 1601G>A transition in exon 10 of the F5 gene on Ch1q23 - p. Arg506Gln in Factor V (R506Q) - (variant known as FV Leiden) - Pro-coagulant activity of FV is limited by Activated Protein C (APC) which cleaves at the arginine (R) - FV Leiden resistant to APC cleavage (APC could no longer inhibit FV) o Excess conversion of prothrombin to thrombin - What category of mutation is this? Mis-sense - FV Leiden allele frequency ~5% in Caucasians (founder effect?) - Heterozygotes 6 – 7 fold increase in relative risk of venous thromboembolism - Homozygotes up to 80-fold increase in relative risk of thromboembolism - Further increased risk of TE disease with Oral Contraceptives – Why? Oestrogen – increases conc. of different factors Warfarin Anticoagulant - Vitamin K antagonist - Used to manage patients at risk of thromboembolic disease - Too much warfarin can be associated with life threatening hemorrhage - Monitor with coagulation tests (PT/INR) - Adjust dose to maintain within therapeutic window Problem 1 You are treating a patient at risk of thromboembolism. The PT/INR is lower than normal. Would you give warfarin? PT/INR measures time taken for blood to clot in this patient compared to a normal persons. PT/INR is lower than normal = too much clotting factor – clotting faster. So yes, would give warfarin Problem 2 The patient has a CYP2C9*2 allele which inhibits warfarin metabolism. Are they likely to need more or less warfarin than usual? CYP450 system allele – gets rid of drugs from the body – warfarin will accumulate because not being metabolised – more warfarin in blood – higher anticoagulation effect – risk of bleeding Things to remember 1. Factor V Leiden is a relatively common inherited tendency to develop venous thrombosis (Ch1) 2. VWD is a very prevalent, but usually mild, heritable bleeding disorder characterized by prolonged bleeding time. It is associated with heterogeneous quantitative or qualitative defects of VWF and inheritance can be dominant or recessive 3. Pharmacogenetics relates to the idea of targeting drug therapy to maximise benefits and limit adverse effects based on individual genetic profile 4. Optimal dose of warfarin (anticoagulant) is related to genotype (CYP2C9 alleles) MD210 Genetics Flipped Lecture 9 – Cystic Fibrosis and Population Screening Puzzle - Why do some communities/ethnic groups have different risk of inherited genetic disorders than the general population? - Founder Effect - reduction in genetic variation due to migration/isolation of a small number of endogamous individuals from a large population Groups That Practice Extensive Endogamy - “marriage” within a specific community - May be a conscious choice to preserve cultural/religious identity - May be choice related to perceived superior social status (Royal Families of Europe) - May be related to social exclusion/caste so that intermarriage is prohibited - In parts of India “Dalit” (official term “Scheduled Castes”) - Japan (“Buraku”) - Nearer to home – some Irish travellers Founder Effect Example - Pennsylvania Amish & Ellis van Creveld Syndrome - Ellis van Creveld: short stature, polydactyly, abnormality of nails and dentition, cardiac defects - Allele frequency 7% Pennsylvania Amish and 0.1% in General Northern European Population - EVC Gene Short Arm of Chromosome 4 The Amish Founder Effect - Relatively discrete breeding sub-population (endogamy) - Derived from a small group of related people (founders) - If a specific mutation was present by chance in the founders it may be disproportionately common in the expanded group derived from that population - Likewise frequent disease associated alleles that are by chance missing in the founders may be disproportionally uncommon in the expanded group derived from that population - Exogamy (marrying unrelated people) will tend to dilute or diminish founder effect Founder Effect and Genetic Drift Fanconi Aplastic Anaemia - FA is a rare genetic syndrome characterized by short stature, various congenital abnormalities, bone marrow failure, and cancer predisposition - Autosomal recessive - Heterozygotes may have short stature, but no bone marrow aplasia or increased cancer risk* - At least 15 genetic variants associated with distinct disease profiles. - 3 important ones: o FANCA Chr 16 o FANCC Chr 9q o FANCG Chr 9p Fanconi Anaemia - FA pathway is responsible for fixing DNA damage during DNA replication - Quick dividing cells most affected – bone marrow, foetal - Predisposition to Malignancy in surviving cells - About 20% homozygotes develop cancer - Median age of onset 16 - Haematological malignancy (AML, MDS, ALL) - Squamous cell cancers - Hepatomas Fanconi Anaemia - Overall prevalence of 1 to 5 per million - Carrier frequency of 1 in 200 to 1 in 300 Founder effects – carrier frequency: - Afrikaner population of South Africa 1 / 77 - Ashkenazi Jewish 1 / 89 - Spanish Gypsies 1 in 64 to 1 in 70 Nearer to home: Some Irish Travelers have increased frequency Ashkenazi Jews - Middle Ages- Jews Living Along the River Rhine - Subsequently centred in Poland and Lithuania - Distinctive customs from Sephardic Jews (little intermarriage – with Sephardic Jews or nonJews) - Recovered from a genetic bottleneck of ~400 families (circa year 1000) (only around 400 families remained and because they were endogamous this led to a decrease in genetic diversity) - Migration to US from late 17th/early 18th century - Relatively small initial population (founders) Ashkenazi Jews - Current Ashkenazi Jewish population in the US (6-7 million) derived from a limited number of people with extensive intermarriage - Relatively high incidence of a number of inherited genetic diseases: (and lower incidence of some other genetic diseases) Cystic Fibrosis (carrier 1/24) Tay-Sachs Disease (carrier 1/25) Canavan Disease (carrier 1/40) Niemann-Pick Disease – Type A (1/90) Gaucher Disease – Type 1 (1/14) Familial Dysautonomia (FD) (1/30) Bloom Syndrome (1/100) Fanconi anaemia – Type C (1/89) Mucolipidosis IV (ML IV) Tay Sachs Carrier Screening - Carrier screening: testing a target population to identify unaffected carriers of a disease allele - Fatal disease in homozygotes - Deficiency in lysosomal enzyme β-hexosaminidase A (HEX A) - substrate GM2 ganglioside accumulates - Blindness, seizures, hypotonia - Death by 5 years - Carrier screening in N. America - Education on risks, testing and reproductive options - 90% reduction in Tay-Sachs disease births between 1970s1990s - 0% by 2003 Principles of Population Screening Each screening programme needs to be appraised for viability, effectiveness and appropriateness in each target population National Screening Committee UK international criteria: - The condition – serious, well understood and relatively common (cost/benefit) - The test – acceptable, easy and cheap, valid and reliable - The intervention – effective treatment/counselling, prenatal diagnosis and ART available - The screening programme –effective (clinical data), ethical, benefit outweighs harms - Implementation criteria – accessibility, resources for diagnosis and treatment, communication of results, data privacy, quality assurance Cystic Fibrosis - CFTR Gene - Cystic Fibrosis Transmembrane Regulator - Ion Channel (Cl- and HCO3-) - Chr 7q31.2 (locus) - 27 Exons & 230 000 base pairs - 12 Transmembrane domains - 2 nucleotide binding domains - 1 regulatory domain - Function: “Gating” - ATP dependent cycling between conducting ions (open) and not conducting ions (closed) Cystic Fibrosis Manifestations – Viscous Secretions - Sweat (Na+ >60 mmol/L) - Lungs – thick sticky mucus, infections - GIT (may present with obstruction in infancy – meconium ileus) - Frequent greasy, bulky stools (steatorrhea) - Sinuses - polyps, thick sticky mucus, infections - Pancreas (malabsorption, failure to thrive, CF) - Male infertility – CBAVD (95% of males and 20% females infertile) Respiratory Mucosa – Normal vs CF Cystic Fibrosis Mutations - Hypomorphic (less function) heterogeneity: thousands of alleles associated with disease are known - Loss of function mutations tend to be more heterogeneous than gain of function mutations - Recessive pattern of inheritance (affected people usually have unaffected parents) - CFTR has biallelic expression - limited/no clinical effect in heterozygote as CFTR protein is haplosufficient Abnormal CFTR Alleles - Abnormal allele in 1/25 of Northern Europeans [1/500 Asians] - Carrier rate in Ireland 1 in 19 - Most common is p.F508del (a.k.a. Delta-F508, ΔF508, DF508) - 70% of CF alleles in Northern Europeans - Deletion of phenylalanine at position 508 in CFTR (in NBD-1) - Altered CFTR protein is degraded intracellularly and does not make it to the apical cell membrane (loss of function) - ΔF508 = Frameshift mutation - Deletion of 3 Nucleotides in Exon 10 - ATC-ATC-TTT-GGT-GTT (wt) - Ile Ile Phe Gly Val - ATC-ATT-GGT-GTT (CF) - Ile Ile Gly Val Mutations in CFTR Gene The frequency of DeltaF508 Depends on Geography - 88% of abnormal alleles in Denmark are DF508 - 50% in Italy - About 30% in Turkey Why is this allele so common ? - Hypothesis: heterozygote advantage (protected against dehydration due to diarrhea – cholera, typhoid fever epidemics) Is Screening Available for Cystic Fibrosis? - Individuals / Couples could be tested to determine if heterozygous (Aa) with a view to planning pregnancy - Testing of embryos for purposes of embryo selection - Prenatal testing on amniotic fluid with a view to termination of pregnancy - Screening of newborns to provide early detection and intervention - The challenge is > 2000 mutations - Easier way to test – blood spot test – blood level of immuno reactive tripsinogen IRT - 99% effective Things to Remember 1. A discrete population for purposes of genetics is defined in terms of mating not geography 2. Founder Effect can result in a high frequency of a specific allele variant in a specific population (e.g Fanconis anaemia in Spanish Gypsies, Tay-Sachs disease in Ashkenazi Jews) 3. Population screening has important ethical considerations and any new screening programme must be appraised for its suitability in each target population 4. Something that is a useful diagnostic test is not necessarily a good population screening test 5. Cystic fibrosis is an autosomal recessive condition common in people of northern European extraction 6. Frame Shift Mutation refers to a mutation that puts the trinucleotide sequences that code for an amino acid out of order Question: An endogamous Ashkenazi Jewish population in America have an increased frequency of a mutated allele for FANCC relative to the general population. Which of the following statements is most appropriate? - It is likely that the practice of endogamy by the Ashkenazi Jews was the only cause for this increased frequency - The frequency of this allele in the general population will likely change more than in the Ashekanzi Jews as a result of genetic drift - It is likely that the founder population of immigrating Ashkenazi Jews had a higher frequency of this mutation than the general population ?? - Around 50% of homozygotes for this mutation will develop cancer Genetics Lecture 9 – Cystic Fibrosis& Genetic Screening Case – Joe - 2nd child - Older brother aged 4 big for age and healthy - Infancy – he is slow to gain weight - Coughs a lot - Hospital admission at 5 months LRTI (lower respiratory tract infection) - Bulky offensive stool - Below 5th Centile for Height and Weight - Sweat Test: Na+ 87mmol/L (upper limit of normal is 60mmol/L) (Sweat test for cystic fibrosis) - What are the implications for cystic fibrosis? Mucus build up, respiratory distress, infertility (95%) - Cystic fibrosis – life impact - Marked reduction in life expectancy - Duration and Quality of Life critically dependent on quality of services - The Biggest Problem: Repeated LRTI with progressive destruction of lung tissue (bronchiectasis and respiratory failure) CF Pulmonary Infection - Major determinant of life expectancy - Different bacteria cause infection at different stages - Age at which they become permanently colonised/infected with Pseudomonas aeruginosa is a critical issue - Where does the bacteria come from? Everywhere – environmental – difficult to prevent exposure Meconium Ileus - Obstruction of the GIT of the infant related to inspissated (thick, dehydrated) material - Occurs in 15 to 20% of infants with CF - Genotype at Cystic Fibrosis Modifier 1 (CFM1) gene on Ch 19 may determine risk of developing meconium ileus Question: You are pretty sure that Joe has CF, how should you confirm the diagnosis? - Define the mutations (targeted mutation panel) - If not a common mutation, what then? Scan exons by PCR amplification and Single Strand Conformation Polymorphism – sequence exons that look different from controls CF Mutation Nomenclature - Cystic Fibrosis Carrier = Aa - A – any CFTR allele that results in a functioning chloride channel (sequence may vary) - a – any CFTR allele that does not code for a functioning chloride channel - (> 2000 mutated CFTR alleles have been described) Mutation categories (Class I to V) include: I. Protein production - (no functional protein produced) II. Protein processing (misfolding) III. Gating (doesn’t open) IV. Conduction (faulty channel) V. Insufficient protein (splice site) Why is diagnosing the mutations so important for Joe? Treatment CFTR Pharmacogenetics - CFTR modulator therapies are designed to correct the malfunctioning protein made by a mutated CFTR: o Read-through compounds (non-sense) o Correctors (misfolding) o Potentiators (open channel/increase function) Some Less Common CF Mutations - Gating Mutations - e.g. G551D (Glycine changed to Aspartic Acid at position 551) - 4-5% of Cases of CF (depends on population) - The CFTR protein is in place in cell membrane but does not work because the chloride channel does not open - Ivacaftor (Kalydeco) (potentiator) binds to CFTR and allows it to open - Does not work for DF508 but combination drug Orkambi (corrector) does New Triple Therapy approved 2020- Kaftrio - Available for patients 12 + with at least one (or two) DF508 and one “minimal unction” mutation since Oct 2020 - Elexacaftor, Tezacaftor (“correctors”) and Ivacaftor (“potentiator”) - Designed to increase the quantity and function of the F508del-CFTR protein at the cell surface - Since March 2023 extended to patients aged 6-11, possibly to 2-5-year-olds in 2024 (pending EMA approval) - Ireland is among the first 4 countries in Europe to secure full access to Kaftrio. (UK, Germany and Denmark) Question: What are the implications for Joe’s extended family? - Carrier analysis may be requested - “Cascade screening/cascade testing” - Carrier testing is available to adults over the age of 16 where there is a family history of CF, or where a family member/partner has been found to carry a CF mutation - Testing is available through the Molecular Genetics Department at the National Centre for Medical Genetics (NCMG), Our Lady's Children’s Hospital Crumlin Cystic Fibrosis Pedigree (doesn’t show carriers) Purposes of Genetic Screening for Cystic Fibrosis - Carrier screening - to plan pregnancy - Testing of embryos - embryo selection - Prenatal testing on amniotic fluid/CVS - termination of pregnancy - Newborn screening - early detection and intervention: o high energy diet, physio, medicines for lung function & gut absorption Newborn Screening for Metabolic Disease Established in 1966 - Guthrie test, “Heel prick test” - CF 1: 1,500 [2011] - Phenylketonuria 1: 4,500 [1966] - Homocystinuria 1: 65,000 [1971] - Classical Galactosaemia 1: 19,000 [1972] - Maple Syrup Urine Disease 1: 125,000 [1972] - Congenital Hypothyroidism 1: 3,500 [1979] - Glutaric acidiuria type 1 (GA1) 1: 54,000 [2018] - MCADD (medium-chain acyl-CoA dehydrogenase deficiency) 1: 66,000 [2018] New-born screening for CF - IRT screening has low specificity (increased IRT in CF) - Relatively high false positive rate - Combined with mutational analysis vastly improves specificity - ~2% of carriers of CF born each year will also be detected Why no CF population carrier screening? - The condition – serious, well understood and relatively common (cost/benefit) (YES) - The test – acceptable, easy and cheap, valid and reliable (NO) - The intervention – effective treatment/counselling, prenatal diagnosis and ART available (YES) - The screening program – effective (clinical data), ethical, benefit outweigh harms (NO) - Implementation criteria – accessibility, resources for diagnosis and treatment, communication of results, data privacy, quality assurance (NO) Bottom line – complementary approach works best – miss fewer infants and less ethical concerns Screening Adults for Carriage - Testing just for DF508 is straightforward - Amplify relevant sequence from genomic DNA - Assess for wt or DF508 sequence (restriction enzyme/oliognucleotide probe) - Asses for DF508 during amplification using real time approach - Other common mutations – 38 mutation panel detects ~93.5% of the CF mutations found in the Irish population - But what about the other >1900? - What can you do if none are detected? Sequencing NGS & Cystic Fibrosis - Despite extensive screening, 1–5% of CF patients lack a definite molecular diagnosis. - Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions – ~99% detection rate - NGS also being used to design custom CFTR mutation panels for different geographic regions, with around 95% detection rates Screening for Carriage - Which mutations to screen for depends on who you are looking at (ethnic background) - In non-Hispanic US Caucasians - ∆F508 = ~70% carriage rate - In US Hispanics = ~46% - The next 5 most common are G542X, G551D, 621+1G>T, W1282X and N1303K - In US Ashkenazi Jews (carriage rate of 1/25) o W1282X (45.92%) o ΔF508 (31.41%), o G542X (7.55%) o 3842+10kbC>T (4.77%) o N1303K (2.78%) - Why is DF508 not the predominant mutation? Genetic drift and founder effect Haemochromatosis - Clinical condition characterised by accumulation of Iron (liver, skin and other tissues) - Clinical manifestations develop in adult life (age of onset very variable) - Hepatic failure, Cardiac failure - Skin pigmentation - Joint Disease - It is relatively common - Diagnosis: o Elevated transferrin saturation o Elevated serum ferritin levels - Treatment – phlebotomy (More common in males – females menstruate) - - Classical Haemochromatosis is associated with variant alleles of the HFE gene 6p21.3 Autosomal recessive pattern HFE regulates iron absorption from the diet and iron storage Deficiency = iron overload 2 key mutations: 1. G to A transition at nucleotide 845 (c.845G>A) - Cysteine to Tyrosine (p.C282Y) 2. C to G at nucleotide 187 (c.187C>G) - Histidine to Aspartic acid 63 (p.H63D) C282Y homozygous in 80-85% hemochromatosis cases (3x increase iron absorption) Also C282Y and H63D Compound Heterozygotes Homozygous H63D does not result in clinically manifest haemochromatosis Penetrance may be as low as 1%, even in homozygotes Is haemochromatosis a hereditary disease? Outcome critically dependent on lifestyle factors “exposome” (e.g. red meat diet vs vegan diet) Should there be a screening program? No – very low penetrance – no actionable knowledge Population Screening - Something that is a useful diagnostic test is not automatically a good population screening test - Likelihood of discovering undiagnosed patient with HH is <1 in 1000 - No evidence of clinical benefit for treatment of asymptomatic carriers - Contraindications for screening: o Low positive predictive value – e.g. Haemochromatosis H63D o Low population attributable risk (PAR) – the proportion of total disease risk in the population attributable to the factor being screened for – e.g. G6PDD mutations o Low absolute risk – e.g. FV Leiden thromboembolism relative risk for oral contraceptive users high, but absolute risk low as most of these are young people o No actionable knowledge – no way to improve prognosis Principles of Screening CDC's ACCE framework: - Analytical validity – how accurate is measurement? - Clinical validity – how accurately does it predict presence/absence of disease? - Clinical utility – how useful are the results (clinical benefit)? - Ethical, legal and social implications? Screening is not a diagnosis – need a specific diagnosis for treatment Screening is only good if can do something with the information Things to remember: 1. Screening is not diagnosis and you need to confirm the diagnosis. 2. Genetic drift is the change in allele frequencies in a population from one generation to the next due to chance. These changes are more pronounced in smaller populations than larger populations. 3. Founder effect can cause some rare heritable disorders to occur with greater frequency, but can also lead to a lower incidence of common heritable disorders in the founder population 4. Haemochromatosis is a good example of a genome based predisposition to disease with outcome critically dependent on “exposome” 5. Contraindications for screening include a low positive predictive value, low population attributable risk and lack of clinical utility Genetics Flipped lesson 10 – Cancer and Genomic Medicine Cancer Terms - Neoplasm is an abnormal mass of tissue unresponsive to normal growth controls - Neoplastic cells have clonally expanded as a result of somatic mutation (somatic mutation – one that occurs at some point during the life of that cell – not heritable) - Benign – uncontrolled growth of tumour, but cannot metastasize - Malignant tumour (cancer) cells have acquired ability to metastasise (seed other places - spread) Cell Growth/Division (Cancer is closely linked to the cell cycle – most cancers have inactivating mutations in one or more proteins that normally function to restrict progression through the G1 stage of the cell cycle. Virtually all human tumours have inactivating mutations in proteins such as P53 that normally function at crucial cell cycle checkpoints, stopping the cycle if a previous step has occurred incorrectly, or if DNA has been damaged. The categories of these proteins are growth factors, growth factor receptors, signal transduction proteins, transcription factors and cell cycle control proteins) Hallmarks of Cancer (Cancer is a progressive pathology – doesn’t happen overnight. Changes accumulate in cancer cell – hallmarks) Some Cancer Related Genes - Protooncogene (Oncogene(- a proto-oncogene that has mutated to become tumoragenic)) – the accelerator: spped up cancer process - Genes typically involved in growth, proliferation, cell cycle o Tyrosine kinases (Src, ABL) o Growth factors (PDGF-B), o Receptor tyrosine kinases (ERBB2, EGFR, HER2/neu) o Intracellular signalling cascade components (Ras) o Cell cycle regulators (Myc) Some Cancer Related Genes - Tumour Suppressor Genes (TSG) – the brakes: slow down progression - “gatekeeper genes” (e.g. APC, RB1) o inhibit tumour growth, proliferation or cell cycle progression - “caretaker genes” (stability genes e.g. MLH1, MSH2) o stabilise the genome (provide mutational (DNA repair) or chromosomal stability) Protooncogene – Oncogene - Oncogene – a gene that when expressed confers resistance to programmed cell death (accelerates progression) - Proto-oncogene – can become an oncogene following: o point mutation o chromosomal translocation o increase in gene expression (e.g. change in promoter) - Change in just one of the two alleles (of a proto-oncogene to turn it into an oncogene) may result in malignant transformation e.g. epidermal growth factor (EGFR) - Dominant activating/hypermorphic (gain of function) mutation Tumour Suppressor Genes - Products of this family of genes regulate cell cycle or direct cells towards apoptosis – code for: 1. Proteins that repress expression of genes essential for continuation of cell cycle 2. Proteins that prevent cell cycle progre