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Genetics for Epidemiologists Lecture 2: Measurement of Genetic Exposures

This lecture covers the measurement of genetic variation, including blood group markers, restriction-fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), single nucleotide polymorphisms (SNPs), linkage disequilibrium (LD), and familial resemblance and family history. Topics are discussed in relation to epidemiology and population genomics.

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Genetics for Epidemiologists Lecture 2: Measurement of Genetic Exposures

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  1. Genetics for EpidemiologistsLecture 2: Measurement of Genetic Exposures National Human Genome Research Institute U.S. Department of Health and Human Services National Institutes of Health National Human Genome Research Institute National Institutes of Health Teri A. Manolio, M.D., Ph.D.Director, Office of Population Genomics and Senior Advisor to the Director, NHGRI, for Population Genomics U.S. Department of Health and Human Services

  2. Topics to be Covered • Measuring genetic variation • Blood group markers • Restriction-fragment length polymorphisms • Variable number of tandem repeats (VNTRs, minisatellites and microsatellites) • Single nucleotide polymorphisms (SNPs) • Linkage disequilibrium (LD) • Familial resemblance and family history

  3. Larson, G. The Complete Far Side. 2003.

  4. Measuring Genetic Variation: Blood Group and Enzymatic Markers • RBC COMT activity measured in 5 large families with hypertension (total 518 individuals) • Associations tested with 25 genetic markers: ABO, Rh, K, MNS,P, Fy, Jk, PGD, ADA, ACP1, PGM1, HBB, GPT, C3, HPA, TF, GC, OR, GM, KM, BF, ESD, GLO1, Le • Lod score of 1.27 and estimated recombination fraction of 0.1 found for phosphogluconate dehydrogenase (PGD) Am J Med Genet 1984; 19:525-32.

  5. Restriction Fragment Length Polymorphisms (RFLPs) • Define polymorphic marker loci that can be detected as differences in length of DNA fragments after digestion with DNA sequence-specific endonucleases • Establish linkage relationships using pedigree analysis Am J Hum Genet 1980; 32:314-331.

  6. Restriction Fragment Length Polymorphisms (RFLPs) Since the RFLPs are being used simply as genetic markers, any trait… segregating in a pedigree can be mapped. Such a procedure would not require any knowledge of the biochemical nature of the trait or of the nature of the alterations in the DNA responsible for the trait. Am J Hum Genet 1980; 32:314-331.

  7. RFLPs Used to Map Neurofibromatosis Linkage analysis of 15 Utah kindreds showed that a gene responsible for von Recklinghausen neurofibromatosis (NF) is located near the centromere on chromosome 17 Science 1987; 236:1100-1102.

  8. RFLPs Used to Map Neurofibromatosis Cosegration of NF with the A2 (1.9 kb) allele and not A1 (2.4kb) in each of four affected offspring. Science 1987; 236:1100-1102.

  9. Variable Numbers of Tandem Repeats (VNTRs): Minisatellites • Repetition in tandem of a short (6- to 100-bp) motif spanning 0.5 kb to several kb • Opened the way to DNA fingerprinting for individual identification • Provided the first highly polymorphic, multiallelic markers for linkage studies • Associated with many interesting features of human genome biology and evolution • Well-known minisatellite is 5.5kb, kringle IV repeat in apolipoprotein(a) and plasminogen Vernaud G and Denoued F, Genome Res 2000; 10:899-907.

  10. Kringle-IV Encoding Sequences of Human apo(a) cDNA ApoA1 Alleles Lackner et al, Hum Mol Genet 1993; 2:933-40.

  11. Correlations of ApoA Molecular Weight with Lp(a) Levels and Number of Kringle-IV Repeats Gavish et al, J Clin Invest 1989; 84:2021-27.

  12. Simple Sequence Repeats (also “VNTRs”): Microsatellites Repetition in tandem of a short (2- to 6-bp) motif from 5-5,000 times • Most are di-, tri-, and tetra-nucleotide repeats repeated 20-50 times • Most are highly polymorphic making them enormously useful for mapping and linkage • Marshfield and similar maps placed ~400 microsatellites across genome, provided primers for analysis • Could be highly automated: NHLBI and CIDR large-scale genotyping services

  13. Multipoint LOD Scores for Long-term SBP and DBP on Chromosome 17 Levy et al, Hypertension 2000;36:477-483.

  14. Larson, G. The Complete Far Side. 2003.

  15. Single Nucleotide Polymorphisms (SNPs) GAAATAATTAATGTTTTCCTTCCTTCTCCTATTTTGTCCTTTACTTCAATTTATTTATTTATTATTAATATTATTATTTTTTGAGACGGAGTTTC/ACTCTTGTTGCCAACCTGGAGTGCAGTGGCGTGATCTCAGCTCACTGCACACTCCGCTTTCCTGGTTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGTCACACACCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGTTGGGGTTTCACCATGTTGGCCAGACTGGTCTCGAACTCCTGACCTTGTGATCCGCCAGCCTCTGCCTCCCAAAGAGCTGGGATTACAGGCGTGAGCCACCGCGCTCGGCCCTTTGCATCAATTTCTACAGCTTGTTTTCTTTGCCTGGACTTTACAAGTCTTACCTTGTTCTGCC/TTCAGATATTTGTGTGGTCTCATTCTGGTGTGCCAGTAGCTAAAAATCCATGATTTGCTCTCATCCCACTCCTGTTGTTCATCTCCTCTTATCTGGGGTCACA/CTATCTCTTCGTGATTGCATTCTGATCCCCAGTACTTAGCATGTGCGTAACAACTCTGCCTCTGCTTTCCCAGGCTGTTGATGGGGTGCTGTTCATGCCTCAGAAAAATGCATTGTAAGTTAAATTATTAAAGATTTTAAATATAGGAAAAAAGTAAGCAAACATAAGGAACAAAAAGGAAAGAACATGTATTCTAATCCATTATTTATTATACAATTAAGAAATTTGGAAACTTTAGATTACACTGCTTTTAGAGATGGAGATGTAGTAAGTCTTTTACTCTTTACAAAATACATGTGTTAGCAATTTTGGGAAGAATAGTAACTCACCCGAACAGTG/TAATGTGAATATGTCACTTACTAGAGGAAAGAAGGCACTTGAAAAACATCTCTAAACCGTATAAAAACAATTACATCATAATGATGAAAACCCAAGGAATTTTTTTAGAAAACATTACCAGGGCTAATAACAAAGTAGAGCCACATGTCATTTATCTTCCCTTTGTGTCTGTGTGAGAATTCTAGAGTTATATTTGTACATAGCATGGAAAAATGAGAGGCTAGTTTATCAACTAGTTCATTTTTAAAAGTCTAACACATCCTAGGTATAGGTGAACTGTCCTCCTGCCAATGTATTGCACATTTGTGCCCAGATCCAGCATAGGGTATGTTTGCCATTTACAAACGTTTATGTCTTAAGAGAGGAAATATGAAGAGCAAAACAGTGCATGCTGGAGAGAGAAAGCTGATACAAATATAAAT/GAAACAATAATTGGAAAAATTGAGAAACTACTCATTTTCTAAATTACTCATGTATTTTCCTAGAATTTAAGTCTTTTAATTTTTGATAAATCCCAATGTGAGACAAGATAAGTATTAGTGATGGTATGAGTAATTAATATCTGTTATATAATATTCATTTTCATAGTGGAAGAAATAAAATAAAGGTTGTGATGATTGTTGATTATTTTTTCTAGAGGGGTTGTCAGGGAAAGAAATTGCTTTTT SNPs 1 / 300 bases ~ 10 million across genome

  16. Mapping the Relationships Among SNPs Christensen and Murray, N Engl J Med 2007; 356:1094-1097.

  17. Chromosome 9p21 Region Associated with MI Samani N et al, N Engl J Med 2007; 357:443-453.

  18. Distances Among East Coast Cities

  19. Distances Among East Coast Cities

  20. Distances Among East Coast Cities

  21. Provi- dence New York Phila- delphia Balti- more Wash- ington Boston Distances Among East Coast Cities

  22. Distances Among East Coast Cities

  23. } One Tag SNP May Serve as Proxy for Many } Block 1 Block 2 CAGATCGCTGGATGAATCGCATCTGTAAGCAT CGGATTGCTGCATGGATCGCATCTGTAAGCAC CAGATCGCTGGATGAATCGCATCTGTAAGCAT CAGATCGCTGGATGAATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAC SNP4 ↓ SNP3 ↓ SNP5 ↓ SNP6 ↓ SNP7 ↓ SNP8 ↓ SNP2 ↓ SNP1 ↓

  24. } One Tag SNP May Serve as Proxy for Many } Block 1 Block 2 CAGATCGCTGGATGAATCGCATCTGTAAGCAT CGGATTGCTGCATGGATCGCATCTGTAAGCAC CAGATCGCTGGATGAATCGCATCTGTAAGCAT CAGATCGCTGGATGAATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAC % SNP4 ↓ SNP3 ↓ SNP5 ↓ SNP6 ↓ SNP7 ↓ SNP8 ↓ SNP2 ↓ SNP1 ↓

  25. } One Tag SNP May Serve as Proxy for Many } Block 1 Block 2 CAGATCGCTGGATGAATCGCATCTGTAAGCAT CGGATTGCTGCATGGATCGCATCTGTAAGCAC CAGATCGCTGGATGAATCGCATCTGTAAGCAT CAGATCGCTGGATGAATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAC % SNP3 ↓ SNP5 ↓ SNP6 ↓ SNP7 ↓ SNP8 ↓

  26. } One Tag SNP May Serve as Proxy for Many } Block 1 Block 2 CAGATCGCTGGATGAATCGCATCTGTAAGCAT CGGATTGCTGCATGGATCGCATCTGTAAGCAC CAGATCGCTGGATGAATCGCATCTGTAAGCAT CAGATCGCTGGATGAATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAT CGGATTGCTGCATGGATCCCATCAGTACGCAC % SNP3 ↓ SNP6 ↓ SNP8 ↓

  27. One Tag SNP May Serve as Proxy for Many Block 1 Block 2 Singleton Frequency GTT 35% CTC 30% GTT 10% GAT 8% CAT 7% CAC 6% other haplotypes 4%

  28. Pair-Wise Linkage Disequilibrium (LD) Measures For a discussion and comparison of these LD measures, see Devlin B, Risch N, Genomics 1995; 29:311-22. Courtesy K. Jacobs, NCI

  29. Two Measures of LD: D' and r2 • D' varies from 0 (complete equilibrium) to 1 (complete disequilibrium) • When D' = 0, typing one SNP provides no information on the other SNP • D' does not adequately account for allele frequencies; r2 is correlation between SNPs, is preferred measure • When r2 = 1, two SNPs are in perfect LD; allele frequencies are identical for both SNPs, and typing one SNP provides complete information on the other

  30. What can LD do for me? • Knowledge of patterns of LD can be quite useful in the design and analysis of genetic data • Design: • Estimation of theoretical power to detect associations • Evaluation of degree of completeness of sampling of genetic variants • Choice of most informative genetic variants to genotype • Sample size increases by ~1/r2 to achieve same power to detect association with SNP2 as SNP1 Courtesy K. Jacobs, NCI

  31. Association Signal for Coronary Artery Disease on Chromosome 9 Samani N et al, N Engl J Med 2007; 357:443-453.

  32. Region of Chromosome 1 Showing Strong Association with Inflammatory Bowel Disease Duerr R et al. Science 2006; 314:1461-63.

  33. LD Patterns in TCF7L2 Association Region Grant et al, Nat Genet 2006; 38:320-23.

  34. LD in Three HapMap Populations International HapMap Consortium, Nature 2005; 437:1299-1320.

  35. A HapMap for More Efficient Association Studies: Goals • Use just the density of SNPs needed to find associations between SNPs and diseases • Do not miss chromosomal regions with disease association • Produce a tool to assist in finding genes affecting health and disease • Ancestral populations differ in their degree of LD; recent African ancestry populations are older and have shorter stretches of LD, need more SNPs for complete genome coverage

  36. SNPs as Gateway to Genome-Wide Association (GWA) Studies • SNPs much more numerous than other markers and easier to assay • Genome-wide studies attempt to capture majority of genomic variation (10M SNPs!) • Variation inherited in groups, or blocks, so not all 10 million points have to be tested • Blocks are shorter (so need to test more points) the less closely people are related • SNP technology allows studies in unrelated persons, assuming 5kb – 10kb lengths in common (300,000 – 1,000,000 markers)

  37. www.hapmap.org International HapMap Consortium, Nature 2005; 437:1299-1320.

  38. www.hapmap.org International HapMap Consortium, Nature 2007; 449:851-861.

  39. Progress in Genotyping Technology 102 ABI TaqMan ABI SNPlex 10 Cost per genotype (Cents, USD) Illumina Golden Gate Affymetrix MegAllele Affymetrix 10K Illumina Infinium/Sentrix Perlegen 1 Affymetrix 100K/500K Nb of SNPs 1 10 102 103 104 105 106 2001 2005 Courtesy S. Chanock, NCI

  40. Continued Progress in Genotyping Technology Affymetrix 500K Illumina 550K Illumina 650Y Illumina 317K Cost per person (USD) July 2005 Oct 2006 Courtesy S. Gabriel, Broad/MIT

  41. Cost of a Genome-Wide Association Study in 2,000 People

  42. Cost of a Genome-Wide Association Study in 2,000 People

  43. Cost of a Genome-Wide Association Study in 2,000 People

  44. Cost of a Genome-Wide Association Study in 2,000 People

  45. Cost of a Genome-Wide Association Study in 2,000 People

  46. Cost of a Genome-Wide Association Study in 2,000 People

  47. Cost of a Genome-Wide Association Study in 2,000 People

  48. Cost of a Genome-Wide Association Study in 2,000 People

  49. Cost of a Genome-Wide Association Study in 2,000 People

  50. Coverage (% SNPs tagged at r2> 0.8) of Commercial Genotyping Platforms Manolio et al, J Clin Invest 2008; 118:1590-605.

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