Genetics - A guide to understanding the literature

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    Notes on slide 1





    Chromosome is an organized structure of DNA - it is one long strand of folded DNA

    Genotype is the genetic constitution of the individual - usually with reference to a specific character in consideration - e.g. Hair color.
    When people talk about the entire genetic constitution of the person it is called genome i.e. It is the complete genetic sequence not just one particular character


    Of ApoE e3 mc in white - e2 & e4 are variant alleles
    Alpha thalassemia and 2 alleles on each chromosome

    Types of polymorphisms/mutation - (insertion/deletion), DNA duplication (copy number variation or CNV), trinucleotide repeat and SNP
    With better understanding - we may just call all alleles as polymorphisms occurring with a particular frequency instead of having the word mutation

    Synonymous SNP - does not lead to change in amino acid sequence
    Non - synonymous SNP - leads to change in amino acid sequence

    Synonymous SNP - does not lead to change in amino acid sequence
    Non synonymous SNP - leads to change in amino acid sequence - the different proteins are called isoforms

    The tendency of alleles to be transmitted together is called linkage. Happens because the alleles are in close proximity

    The alleles themselves are referred to as haplotypes

    Measure of association of the linkage i.e. How often will all the alleles be transmitted together



    A situation in which a defined population displays constant genotype frequencies across multiple generation. The genotype frequency can be calculated using the HW formula






    Evaluates association of genetic variations with outcomes or traits of interest by using 100,000 to million markers across the genome


    Case control studies do not suffer from recall bias as genes do not change





    Investigators may diagnose more people with dementia if they are positive for ApoE e4/e4 allele

    Genetic studies may be misleading if the population studied has mixed ethnicity - specific type of confounding factor in genetic studies.
    Spurious association if particular allele is more common in a particular ethnic group



    In family based controls the SNP with the strongest correlation might be the offending gene


    Sampling error due to taking samples from different sites or taking it at different time
    E.g. Stored blood will have more artificial polymorphisms compared to fresh blood


    Diseased patients may have higher frequency of the allele and thus will not obey HW law
    P-value is obtained through a computer based statistical program







    Choice of calculation depends on relevant biology although it may not be known!!!

    CYP1-5 all predispose to particular disease
    CYP1 is most frequent allele - crosses p-value and the entire risk of CYP1-5 gets attributed to CYP1










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    Genetics - A guide to understanding the literature - Presentation Transcript

    1. GENETICS A guide to understanding the literature Sudeep Bansal Wednesday, March 4, 2009
    2. How to Use an Article About Genetic Association: A: Background Concepts John Attia; John P. A. Ioannidis; Ammarin Thakkinstian; et al. Online article and related content current as of February 3, 2009. JAMA. 2009;301(1):74-81 (doi:10.1001/jama.2008.901) http://jama.ama-assn.org/cgi/content/full/301/1/74 Contact me if this article is corrected. Correction This article has been cited 2 times. Citations How to Use an Article About Genetic Association: B: Contact me when this article is cited. Are the Results of the Study Valid? Topic collections Neurology; Dementias; Neurogenetics; Genetics; Genetic Disorders; Genetics, Other John Attia; John P. A. Ioannidis; Ammarin Thakkinstian; et al. Contact me when new articles are published in these topic areas. Online article and related content current as of February 3, 2009. JAMA. 2009;301(2):191-197 (doi:10.1001/jama.2008.946) http://jama.ama-assn.org/cgi/content/full/301/2/191 Subscribe Email Alerts http://jama.com/subscribe http://jamaarchives.com/alerts Contact me if this article is corrected. CorrectionPermissions Reprints/E-prints permissions@ama-assn.org reprints@ama-assn.org This article has been cited 1 time. Citations http://pubs.ama-assn.org/misc/permissions.dtl How to Use this article is cited. Contact me when an Article About Genetic Association: C: What Are the Results and Will They Alzheimer Disease; Dementias; Journalology/ Peer Review/ Authorship; Neurology; Help Me in Caring Topic collections for My Patients? and Research Methods; Genetics; Genetic Counseling/ Neurogenetics; Statistics Testing/ Therapy; Genetics, Other Online article and related content John Attia; when new A. Ioannidis; Ammarin Thakkinstian; et al. Contact me John P. articles are published in these topic areas. current as of February 3, 2009. JAMA. 2009;301(3):304-308 (doi:10.1001/jama.2008.993) http://jama.ama-assn.org/cgi/content/full/301/3/304 Subscribe Email Alerts http://jama.com/subscribe http://jamaarchives.com/alerts Contact me if this article is corrected. CorrectionPermissions Reprints/E-prints permissions@ama-assn.org reprints@ama-assn.org Citations http://pubs.ama-assn.org/misc/permissions.dtl cited 1 time. This article has been Contact me when this article is cited. Wednesday, March 4, 2009
    3. Box. Glossary Additive Linkage disequilibrium Describes any trait that increases proportionately in expres- A measure of association between alleles at different loci sion when comparing those with no copy, 1 copy, or 2 cop- Locus/loci ies of that allele, ie, those with 1 copy of the allele show more The site(s) on a chromosome at which the gene for a particu- of the trait than those without, and in turn, those with 2 lar trait is located or on a gene at which a particular SNP is copies show more of the trait than those with 1 copy located Allele Messenger RNA One of several variants of a gene, usually referring to a spe- A ribonucleic acid−containing single-strand copy of a gene cific site within the gene that migrates out of the cell nucleus to the ribosome, where Candidate gene study it is translated into a protein A study that evaluates association of specific genetic vari- Mutation ants with outcomes or traits of interest, selecting the vari- A rare variant in a gene, occurring in 1% of a population; ants to be tested according to explicit considerations (known cf polymorphism or postulated biology or function, previous studies, etc) Pedigree Chromosome A diagram depicting heritable traits across 2 or more Self-replicating structures in the nucleus of a cell that carry generations of a family the genetic information Phenotype Dominant The observable characteristics of a cell or organism, usu- Describes any trait that is expressed in a heterozygote, ie, ally being the result of the product coded by a gene (geno- one copy of that allele is sufficient to manifest its effect type) Genome Definitions Polymorphism The entire collection of genetic information (or genes) that The existence of 2 or more variants of a gene, occurring in an organism possesses a population, with at least 1% frequency of the less com- Genome-wide association study mon variant (cf mutation) A study that evaluates association of genetic variation with Recessive outcomes or traits of interest by using 100 000 to 1 000 000 Describes any trait that is expressed in a homozygote but or more markers across the genome not a heterozygote, ie, 2 copies of that allele are necessary Genotype to manifest its effect The genetic constitution of an individual, either overall or Ribosome at a specific gene The protein synthesis machinery of a cell where messenger Haplotype RNA translation occurs Alleles that tend to occur together on the same chromo- SNP some because of SNPs being in proximity and therefore in- Abbreviation for single-nucleotide polymorphism, a herited together single base pair change in the DNA sequence at a particu- Heterozygous lar point compared with the “common” or “wild-type” An individual is heterozygous at a gene location if he or she sequence has 2 different alleles (one on the maternal chromosome and Synonymous SNP one on the paternal) at that location A SNP that does not lead to a change in the amino acid Homozygous sequence compared with the common or wild-type An individual is homozygous at a gene location if he or she sequence; cf nonsynonymous, in which there is a change has 2 identical alleles at that location in the amino acid sequence as a result of the SNP Isoform Variant allele Variant in the amino acid sequence of a protein The allele at a particular SNP that is the least frequent in a population Linkage Wild-type allele The tendency of genes or other DNA sequences at specific loci to be inherited together as a consequence of their physi- The allele at a particular SNP that is most frequent in a popu- cal proximity on a single chromosome lation, also called “common” allele Wednesday, March 4, 2009 ©2009 American Medical Association. All rights reserved. 75 (Reprinted) JAMA, January 7, 2009—Vol 301, No. 1
    4. { Protein Gene Wednesday, March 4, 2009
    5. Genotype & Phenotype Wednesday, March 4, 2009
    6. Variant gene for red Variant gene for blue color color Homologous Chromosomes Allele Wednesday, March 4, 2009
    7. Two alleles on { same chromosome Allele Wednesday, March 4, 2009
    8. Variant of Gene Is it > 1% Yes No Mutation Polymorphism The other 99% is called “Wild type” or Common allele The least frequent gene variant is called Variant allele Wednesday, March 4, 2009
    9. Single Nucleotide Polymorphism (SNP) Synonymous Non Synonymous Wednesday, March 4, 2009
    10. Single Nucleotide Polymorphism (SNP) Synonymous Non Synonymous Wednesday, March 4, 2009
    11. Meiosis Linkage Wednesday, March 4, 2009
    12. Two alleles on { same chromosome Meiosis Haplotype Wednesday, March 4, 2009
    13. Lost gene Replication Linkage disequilibrium Wednesday, March 4, 2009
    14. Homozygous Heterozygous Additive/Per-Allele Blue is Red is Red & Blue are autosomal recessive autosomal dominant Co-dominant Wednesday, March 4, 2009
    15. p+q = p2 + q2 2 pq + =1 Hardy-Weinburg Equilibrium Wednesday, March 4, 2009
    16. Female A (p) a (q) A (p) AA (p2) Aa (pq) Male a (q) aA (pq) aa (q2) p + q = p2 + 2pq + q2 = 1 Hardy-Weinburg Equilibrium Wednesday, March 4, 2009
    17. Deviations from Hardy Weinburg Equilibrium • Inbreeding • Migration • New mutations • Selection disadvantage • Error in studies Wednesday, March 4, 2009
    18. ApoE gene Candidate Gene Study Wednesday, March 4, 2009
    19. Candidate Gene Study Advantages Disadvantages • Targets known genes • Limited to only one gene • Easier to perform & interpret Wednesday, March 4, 2009
    20. Candidate Gene Study Advantages Disadvantages • Targets known genes • Limited to only one gene • Easier to perform & interpret How do you find out which gene to study? Wednesday, March 4, 2009
    21. Genome wide association study Wednesday, March 4, 2009
    22. Genome Wide Association Study Advantages Disadvantages • Targets thousands of genes • Many spurious associations • Prior knowledge not required • Need conformation of findings • Hypothesis generating • Interrogate traditional candidates Wednesday, March 4, 2009
    23. How Are GWA Studies Done? • Traditional methods: • Cohort Studies • Case Control Wednesday, March 4, 2009
    24. Framework To Analyze Literature Wednesday, March 4, 2009
    25. the results of the study Valid • Are are the Results • What is the Clinical Significance of these results • What Wednesday, March 4, 2009
    26. Are the results of the study 1. Valid? Wednesday, March 4, 2009
    27. Which way home? Phenotype definition Wednesday, March 4, 2009
    28. Phenotype measurement & Blinding Wednesday, March 4, 2009
    29. He is demented He has sarcoidosis Ethnic differences and Population Stratification Wednesday, March 4, 2009
    30. GWA Study for Prostate Cancer Population Significant Allele Wednesday, March 4, 2009
    31. GWA Study for Prostate Cancer Population Significant Allele •Red Allele is more common in Blacks •Prostate Cancer is more common in Blacks •Spurious association between red allele & prostate cancer Wednesday, March 4, 2009
    32. Checking For Population Stratification • Self reported ethnicity • Family based controls • Genomic control (testing patterns in unlinked SNP’s) Wednesday, March 4, 2009
    33. Your genetic tests show that you are predisposed to sue doctors! Are the Genetic tests accurate? Wednesday, March 4, 2009
    34. Genotyping Errors • Reasons for error: • Sampling errors • Sample handling • Methodology • Heterozygosity • Rates vary from 1-30% Wednesday, March 4, 2009
    35. “Call Rate” • Proportion of samples with unambiguous reading • Should be > 90-95% Wednesday, March 4, 2009
    36. Does The Study Observe HW Equilibrium • Non-specific & insensitive • Tested only in controls (as they are representative of general population) • 5% of SNP’s will violate HW equilibrium with p-value=0.05 • What if these SNP’s are associated with disease!!!!! Wednesday, March 4, 2009
    37. Significance Of P-value • Consider GWA study testing 100 SNP’s •5 SNP’s may give spurious association for p-value of 0.05 • Statistical probability of one spurious association - 99.4% • Publication bias for positive results !!!!! Wednesday, March 4, 2009
    38. Adjusting P-value • Many methods available • Bonferroni method = p-value / number of SNP’s • E.g. .05/100 = .0005 • For GWA studies > 500,000 SNP’s • P-value = 5x10-8 Wednesday, March 4, 2009
    39. Consistency Of Results • Use Caution - unless results are reproduced • Human Genome Epidemiology Network (HuGE Net) is the cochrane equivalent • NIH genetic studies database Wednesday, March 4, 2009
    40. 2. What are the Results ? Wednesday, March 4, 2009
    41. How Large And Precise Are The Associations • Risk is usually expressed as: • Odds ratio (OR) in case control studies • Relative risk (RR) in cohort studies • Hazard ratio (HR) in survival analysis • Don’t just look for statistical significance • 1.4 fold vs 8 fold increased risk makes a huge difference Wednesday, March 4, 2009
    42. Allele Specific Risk • Dominant allele may result in maximal risk • Recessive allele may result in sub-maximal risk • Presence of 2 recessive alleles will increase risk further • In AR cases, multiple risk ratios may need to be reported for each additional allele present Wednesday, March 4, 2009
    43. Calculating Allele Specific Risk additive or Per allele model = (Risk)No. of alleles • Log additive model = Risk x No. of Alleles • Linear Wednesday, March 4, 2009
    44. Winner’s Curse • GWA overestimates the genetic influence of alleles found to be associated with diseases • Occurs because overestimates are more likely to cross p-value Wednesday, March 4, 2009
    45. 5 fold risk 5 implicated alleles Disease X Wednesday, March 4, 2009
    46. 5 fold risk 5 implicated alleles GWA Study Disease X Most frequent Allele (Winner’s Curse) Wednesday, March 4, 2009
    47. 5 fold risk 5 implicated alleles GWA Study Disease X Does this single allele cause 5 fold risk? Most frequent Allele (Winner’s Curse) Wednesday, March 4, 2009
    48. Genetic Profiles • One gene (with multiple alleles) may have small effect on disease • Combination of multiple genes affecting a disease may increase risk prediction • Beware of gene-gene interactions • Net risk may not be as high as predicted by genetic profile! Wednesday, March 4, 2009
    49. 3. Clinical Significance Wednesday, March 4, 2009
    50. Improved Prediction Beyond Clinical Variables • Certain genes may affect clinical variables • Adjust for family history ! Wednesday, March 4, 2009
    51. Absolute Vs Relative Risk • Factor V Leiden carries 4-5 fold risk for DVT/PE • Risk in population for DVT/PE is ~ 0.2% • Presence of Factor V Leiden in patient with idiopathic DVT/PE will ↑ recurrence risk to almost 30% • Risk of having Factor V Leiden in patients with idiopathic DVT/PE is ~ 12-20% Wednesday, March 4, 2009
    52. Is the risk associated allele present in my patient? Wednesday, March 4, 2009
    53. Is the patient better off knowing his genetic information? Wednesday, March 4, 2009
    54. Summary • GWA study thousands of genes at the same time • Hypothesis generating • Needs follow up with candidate gene studies •? Improvement in prediction beyond established clinical variables Wednesday, March 4, 2009
    55. THANK YOU Wednesday, March 4, 2009
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