Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Quantium Medical Cardiac Output wikipedia , lookup
Cardiac contractility modulation wikipedia , lookup
Electrocardiography wikipedia , lookup
Coronary artery disease wikipedia , lookup
Heart failure wikipedia , lookup
Baker Heart and Diabetes Institute wikipedia , lookup
Arrhythmogenic right ventricular dysplasia wikipedia , lookup
How to find an association between a disease and a gene? Alex V. Postma Department of Anatomy, Embryology & Phsyiology Heart Failure Research Center AMC [email protected] NHS Heart Failure Research Center Sudden cardiac death, the numbers - 50% of cardiac mortality - US: ~250.000 SCD / year - NL: ~300 SCD / week, 15.000 / year - VF most common underlying arrhythmia Heart Failure Research Center Sudden cardiac death, the causes > 40 y 1/1.000/year > 85% coronary artery disease < 40 y 0,8-1,6/100.000/year ~75% genetic disease Cardiomyopathies, primary electrical disease Heart Failure Research Center Monogenic Cardiac Arrhythmia Syndromes Long QT Syndrome Brugada Syndrome Catecholamine-induced PMVT/VF Short QT Syndrome Isolated Cardiac Conduction Disorders Familial Atrial Fibrillation Sinus Node Disease Heart Failure Research Center LQTS, SQTS LQTS, SQTS Sinus Node Dysfunction LQTS, SQTS LQTS BrS CD AF Heart Failure Research Center GENE KCNQ1 KCNH2 KCNE1 KCNE2 KCNJ2 KCNA5 SCN5A SCN4B RYR2 CASQ2 CACNA1C HCN4 GJA5 CAV3 ANKB GPD1L DISORDER LQTS, SQTS, AF LQTS, SQTS LQTS LQTS LQTS, SQTS AF LQTS, BrS, CD, SSS LQTS CPVT CPVT LQTS SSS AF LQTS LQTS BrS Heart Failure Research Center From patient to genotype • Patient goes to cardiologist (complaints, family history,...) • Genetic counseling • DNA isolated • Mutation screening (PCR, sequencing) • Clinical geneticist and cardiologist discuss result / treatment with patient • Family screening OR • Research Heart Failure Research Center Where to start? • Linkage analysis • Screen candidate genes • Perform exome / genome sequencing Heart Failure Research Center Identifying genetic cause for a disease Many human diseases/phenotypes are affected by single genes. Examples include: Brown eyes (dominant to blue eyes); Ability to curl or roll up tongue (dominant to inability) PEDIGREES of large families can help identify whether a genetic disease is caused by a dominant or a recessive allele, and whether the disease is sex-linked or autosomal. Heart Failure Research Center Autosomal Dominant Transmission Heart Failure Research Center Autosomal Dominant Transmission Heart Failure Research Center Autosomal Recessive Transmission Appears in both male and female children of unaffected parents. e.g. KCNQ1 mutations associated with Jervell Lange Nielsen Syndrome Heart Failure Research Center Autosomal Recessive Transmission Appears in both male and female children of unaffected parents. Heart Failure Research Center Mutation Arising De Novo e.g. Timothy Syndrome (CACNAIC) Heart Failure Research Center Mutation Arising De Novo e.g. Timothy Syndrome (CACNAIC) Heart Failure Research Center Compound heterozygosity (multiple variants together) Bezzina et al., Circ Res, 2003 Heart Failure Research Center Linkage analysis Technique to find causative regions / genes for a genetic disease Linkage is co-segregation between a disease and a set of markers >> identify regions that are consistently shared by affected relatives Heart Failure Research Center Genetic markers: • Genetic markers are DNA sequences that show variations in size or sequence in the population • Occur random all over the genome off all species • Probably errors of DNA copying • Inherited to next generations Markers Heart Failure Research Center Cross-over Parents A a B a a b b b Affected First generation A a B a a b b b Affected Second generation A a B Crossover has taken place b Affected A a a a b b b ? a a b B b Affected Heart Failure Research Center Example linkage analysis Heart Failure Research Center Candidate gene approach -Study literature, study disease, hypothesize -Perform linkage analysis (on all candidate genes) -Positive linkage -> look for interesting areas of the gene -Sequence -Find mutation…. Heart Failure Research Center Mutations Mutations are heritable changes in genes or chromosomes. Most mutations are SINGLE-GENE MUTATIONS that arise from errors in replication or from unrepaired damage to DNA molecules. Heart Failure Research Center Types of mutations Original DNA strand Substitution Deletion Insertion Heart Failure Research Center Effect on the protein Silent Missense Nonsense Frameshift Ser Thr Pro Heart Failure Research Center Why mutation screening? • Diagnostic screening: – Confirmation clinical diagnosis – (Prenatal) counseling • Research: – Genotype-phenotype relationship – Pathophysiological mechanisms – Presymptomatic screening/therapy Heart Failure Research Center Mutation Interpretation • Not every novel change leads to disease. • Interpret in conjunction with: – Inheritance pattern – Mechanism of disease – Amino acid conservation – Cis vs. trans – Population/Haplotype information – Structural and functional domains Heart Failure Research Center How to find a mutation • DNA isolation from: Heart Failure Research Center How to find a mutation The Invention of PCR Invented by Kary Mullis in 1983 First published account appeared in 1985 Awarded Nobel Prize for Chemistry in 1993 Heart Failure Research Center Intron-exon structure of a gene Start (ATG) 5’ Stop (TAA, TAG or TGA) AG 3’ GT PCR fragments Heart Failure Research Center What is the Polymerase Chain Reaction? • A means of selectively amplifying a particular segment of DNA • The segment may represent a small part of a large complex mixture of DNAs: e.g. a specific exon of a gene • It can amplify a usable amount of DNA (visible by gel electrophoresis) in ~ 2 hours • It can amplify a single DNA molecule (e.g. from a single sperm) • PCR product usually undergoes post-PCR analysis: restriction digestion, sequencing, cloning etc Heart Failure Research Center Sanger sequencing - DNA fragments are separated by size by gel electrophoresis - From the gel, the DNA sequence can be determined - Can produce DNA fragments 700-900bp long, but low throughput - The Human Genome Project used Sanger sequencing, completion took over 10 years Heart Failure Research Center wildtype A I-1 ATC CAC ATA I H I ATC CRC ATA I H/R I ? Heart Failure Research Center Next Generation Sequencing (NGS) a.k.a. Whole Genome/Exome Sequencing Mega Sequencing 2nd Generation Sequencing Massive parallel Sequencing High Throughput Sequencing Deep Sequencing General characteristics include: - Amplification of genetic material by PCR - Ligation of amplified material to a solid surface - Short reads applications; sequence and then use computers to assemble the small pieces Heart Failure Research Center Next Generation Sequencers Heart Failure Research Center However... • 20K variants identified per exome • ~45% missense, 150 nonsense • 150-250 unique variants / individual • ETHICAL ISSUES!!! Heart Failure Research Center Monogenic vs. polygenic Monogenic disease Polygenic (complex) disease • Severe phenotype • Mild phenotype • Early onset • Late onset • Rare • Common • Mendelian inheritance • Complex inheritance Cause: Mutations (<1%) Polymorphisms (>1%) Many polymorphisms with each a small effect, combined with environmental factors, explain inter-individual differences in risk Heart Failure Research Center Genetic modifiers • The ultimate clinical presentation not only depends on the gene affected or the type / location of the mutation but also on the GENETIC BACKGROUND on which it occurs. • Clinical presentation = effect of mutation + genetic variation in genes (polymorphisms) encoding other players in that particular biological pathway Heart Failure Research Center Polymorphisms ~1/300 bp is polymorphic in a population (3 million loci = 0.1% of the genome) ~1/1200 bp will differ between 2 random subjects Single Nucleotide Polymorphisms (SNPs) are the most common variants Heart Failure Research Center What is the evidence for a role of polymorphisms in susceptibility to sudden cardiac death ? 1. Variability in clinical severity among patients with primary electrical disease 2. Heritability of various ECG parameters 3. Family history Heart Failure Research Center Large family with SCN5A 1795insD mutation Total family members: Unexplained SCD: Nocturnal : SCD <40 yr : 232 26 17 20/26 Total: HR (bpm): PQ (s): QRS (ms): QTc (s): ST-elev V1-V3: ST-elev (mm): Carriers Non-carriers 114 65.4±16.8 118 72.0±12.1 0.20±0.02 115±15 0.48±0.09 21/43 (49%) 1.6±1.0 0.16±0.03 # 93±13 # 0.40±0.03 # 3/36 (8%) # 0.5±0.6 # Bezzina et al., Circ Res 1999;85:1206 van den Berg et. al. JCE. 2001;12:630-6 Heart Failure Research Center Reduced penetrance and variable disease expressivity and severity in patients with SCN5A 1795insD Heart Failure Research Center What is the evidence for a role of polymorphisms in susceptibility to sudden cardiac death ? 1. Variability in clinical severity among patients with primary electrical disease 2. Heritability of various ECG parameters 3. Family history Heart Failure Research Center Common sodium channel promoter haplotype in Asian subjects underlies variability in cardiac conduction T T T --- G C 75.5% Haplotype B C C G ins C T 24% Haplotype C C T T --- C C 0.5% T-847G -835insGC G-354C C287T T-1418C Haplotype A T-1062C Frequency promoter intron 1 exon 1 (non-coding) -2000 bp -1000 0 1000 Bezzina et al., Circulation 2006;113:338-44. Heart Failure Research Center Haplotype B is associated with slower conduction; Haplotype pair effects on PR-interval Brugada syndrome baseline PR (msec) 350 Non-Brugada syndrome controls ** 300 * ** 250 * 200 150 100 BB (5) AB (20) AA (45) BB (8) AB (33) AA (60) *P<0.05; **P≤0.001 Bonferroni-corrected significance level: 0.002 Heart Failure Research Center Reporter activity is reduced for Haplotype B CHO cells Cardiomyocytes n=13, p=0.04 n=9, p=0.006 Fold activity 18 15 12 9 6 3 0 Hap A Hap B Hap A ? Hap B Heart Failure Research Center What is the evidence for a role of polymorphisms in susceptibility to sudden cardiac death ? 1. Variability in clinical severity among patients with primary electrical disease 2. Heritability of various ECG parameters 3. Family history Heart Failure Research Center Family history of sudden death is an important predictor of ventricular fibrillation risk in myocardial infarction MI + VF (cases) Family history of sudden death Male gender Smoking, at time of infarct Hypertension Diabetes Hypercholesterolemia BMI Medication Cardiac history (last 12 mo) Anterior location infarct Center ST deviation versus MI - VF (controls) Univariate OR 95% CI P 2.33 0.72 1.17 0.96 0.58 0.65 0.95 0.99 1.86 1.25 1.65–3.28 0.49–1.05 0.85–1.61 0.69–1.32 0.31–1.06 0.48–0.89 0.91–0.99 0.69–1.43 1.50–3.28 0.92–1.70 1.58 1.30–1.91 <0.0001 0.084 0.34 0.75 0.1 0.007 0.01 0.98 0.033 0.15 0.002 <0.0001 Multivariate OR 95% CI P 2.72 0.72 1.32 1.01 0.64 0.64 0.96 1.04 1.92 1.26 1.84–4.03 0.45–1.14 0.90–1.95 0.64–1.61 0.30–1.35 0.44–0.95 0.91–1.00 0.62–1.74 0.95–3.85 0.86–1.84 1.59 1.25–2.02 <0.0001 0.16 0.16 0.96 0.24 0.027 0.066 0.88 0.068 0.23 0.001 <0.0001 Dekker et al., Circulation 2006; 114: 1140 - 1145 Heart Failure Research Center ASSOCIATION STUDIES How to find the polymorphisms that confer susceptibility to sudden cardiac death? - Large sample sizes - Strict definition of clinical phenotype > same pathophysiological mechanism Heart Failure Research Center Identification of genetic determinants of SCD risk • Association studies in disease populations with the SCD phenotype genetic variation SCD phenotype • Identification of genetic variation associated with specific endophenotyes genetic variation SCD phenotype intermediate phenotypes (endophenotypes) conduction (QRS duration) repolarization (QT interval) - accurate measurement in large sample sizes - quantitative traits; more powerful for genetic analysis Heart Failure Research Center Prolonged QT interval and risk of sudden death in the population Straus et al.,JACC 2006 Heart Failure Research Center Genome-wide association study of QT-interval in the general population [Nitric oxide synthase 1 adaptor protein] Heart Failure Research Center Key points Several cardiac arrhythmia syndromes that for long were considered idiopathic are now known to have a genetic basis The genetic basis for most arrhythmia syndromes is heterogeneous Genetic testing is recommended to support the clinical diagnosis, to identify silent carriers and to install gene-specific therapy Considerable heterogeneity may exist in disease manifestation among family members carrying the same mutation. Future research in genetics of arrhythmias will likely focus on identification of common genetic variation to risk of arrhythmia / sudden cardiac death Heart Failure Research Center Opdrachten • Duo 1: presenteer het artikel van Alders et al. (2009) • Duo 2: presenteer het artikel van Bezzina et al. (2010) • Duo 3: presenteer het artikel van Sotoodehnia et al. (2010) • PowerPoint-presentatie van maximaal 10 minuten • PowerPoint slides in het Engels • Mondelinge presentatie naar keuze in het Nederlands of in het Engels Heart Failure Research Center