Population Genetics of Structural Variation Speaker Dr. Don Conrad

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1 Population Genetics of Structural Variation Dr. Don Conrad Wellcome Trust Sanger Institute Cambridge, UK 1 Overview Ascertainment of CNVs What CNV data is available Humans, chimpanzee, macaque, Drosophila, mouse, rat Population genetic forces: mutation, selection, drift Mutation Processes Rates Recombination/gene conversion Demography/population structure Functional impact of CNVs What are constraints on copy number? Evidence for positive selection Fate of duplicated genes Future challenges/conclusions 2 Available data Human Whole genome surveys in 270 HapMap samples Redon et al., 2006, Nature; McCarroll et al., 2008, Nature Genetics Whole genome survey in 1000 global samples from the HGDP, Jakobsson et al., 2008, Nature Chimpanzee Perry et al., 06 PNAS ; Perry, et al., 08 Genome Research (30 samples) Macaque Lee et al., 08 Genome Research Drosophila Mice Rat Emerson et al., 08 Science, 15 lines of Drosophila Melanogaster Egan et al., 07 Nat Genet Graubert et al., 07 PLoS Genetics Guryev et al., 08 Nat Genet, 14 lines 3 1

2 Ascertainment frequency Power frequency Deletion length (kb) Power to detect a single deletion, as a function of deletion size in HapMap release 16.1c length of deletion (kb) Observed and predicted deletion sizes Conrad et al., (2006) Nature Genetics 4 The forces of population genetics Time The core population genetic forces: Mutation - input of new variation Drift - random change in allele frequency due to sampling Selection - alters the lifetime of mutation (e.g., accelerate the removal or fixation) Key point: all 3 forces are scaled by the population size implications for comparison of CNV patterns across populations and species 5 Mutation-drift-selection balance equency Fre f mutation mutation normal duplication The frequency of an allele at a given point in time results from the balance of the forces of mutation, selection, and drift; mutation adds new copies to the population, while drift and selection can remove them time 6 2

3 Structural variation mutation rates minisatellites Microsatellites CTCT -> CTCTCT Single nucleotide polymorphisms (SNPs) C->A Structural Variation (Deletions, Duplications, Inversions, Translocations) Retroelement insertions Jobling, Hurles, Tyler-Smith, Human Evolutionary Genetics 7 CNV breakpoints - clues to mechanisms Better understanding of mutational mechanisms better mutation models: Predict unobserved pathogenic mutations Power calculations for rare variant studies e.g. combine SNPs, indels and CNVs to maximise power Segmental duplications Density of G-quadraplex Left Flank CNV Right Flank Non-B DNA forming sequences: G-tetraplexes Left Flank CNV Right Flank 8 Mutation rates, cont. NAHR: events per locus/per generation Deletion 2x likely as duplications (D Turner et al., (2008) Nat Genet) Non homologous end joining: <10-7 Fork Stalling and Template Switching (FoSTeS): Rate unknown (Lee and Lupski et al., (2008) Cell) Retroelement insertion: 1 every births; Relative rates: - L1 (6kb): 8.4% - Alu (300bp): 87.2% - HERVK (10kb): 0.5% - SVA (1.6kb): 5% Inter-chromosomal duplication: rate unknown 9 3

4 Mutator alleles Inversion polymorphisms predispose their carrier to further chromosomal rearrangements An inversion is necessary for the CNV to form in some cases: - Sharp et al., (2006) Nat Genet, Visser et al.,.(2005) Am J Hum Genet, Giorda et al., (2007) Hum Mutat, Jobling et al., (1998) Curr Biol In other cases, inversions are enriched in the parents of children with de novo CNVs, but not required for rearrangement: - Gimelli et al., (2003) Hum Mol Genet, Osborne et al., (2001) Nat Genet A Possible mechanisms B centromere Heterologous synapsis Microloop Adapted from AJ Sharp (2008) Hum Mutat inversion telomere Heterologous synapsis Brown et al., (1998) Am J Hum Genet 10 Secondary affects of SV Additional ways in which SV can modulate molecular genetic processes: Large duplications suppress recombination in heterozygous form (Roberts and Broderick (1982) Genetics); Same should be true of large deletions Recombination is restricted between inversions in heterozygous individuals Both of these phenomenon should leave detectable traces in population genetic data a b c Recombination rate (p/kb) Inverted and noninverted Inverted noninverted Physical position (kb) Conrad and Hurles (2007) Nat Genet 11 SNPs and CNVs have similar LD properties C Conrad and Hurles (2007), Nature Genetics McCarroll et al., (2008) Nat Genet 12 4

5 Gene conversion moves variation T A T T Cartoon example of gene conversion between a duplication pair At least 30% of the euchromatic portion of the Y chromosome is contained in palindromic repeats of > 99.9% identity; these repeats also house most of the testis-specific genes on the Y; gene conversion between repeats provides a mechanism for the efficient removal of deleterious variation from the population Palindromes Skaletsky et al., (2003) Nature 13 Size distribution of CNVs >100bp CNV size distributions 2007/2008 Ventor >100 Watson >100 Redon 500K Korbel deletion Kidd deletion Densi ity 2006 Log10(CNV) length) 14 Landscape of CNVs in humans Within an individual appears to be ~10000 variants greater than 50bp in length (Standard array-based platforms are calling CNVs per genome) At the population level: Affymetrix 6.0 (McCarroll et al., 2008 Nat Genet) Synthesis of most CNV studies prior to 2007 Median length of 7.5 kb 1500 CNVs with at least 2 variants in 270 HapMap samples 374 common large CNVs (allele frequency > 5%) in Europeans (CEU) 15 5

6 Population structure and differentiation Conrad and Hurles (2007) Nat Genet CNVs appear quite similar to SNPs in terms of their informativeness for identifying structure, and in terms of their patterns of population differentiation F st for CNPs McCarroll et al., (2008) Nat Genet F st for SNPs 16 Population differentiation V ST =(V T -V S )/V T Vst for 27K BAC clones V ST CNV Genotypes UGT2B17 CCL3L1 Near MAPT Redon et al., 2006 Nature 17 Functional impact of CNVs in humans CNVs explain at least 9-18% of heritable variation in gene expression Suggests that per locus, CNV may affect 5 times as much variation as SNPs Strong prior evidence for an important role of CNV in human traits Stranger et al., (2007) Science 18 6

7 Deletions are deleterious Category CEU deletions YRI deletions Entire HapMap Coding non-synonymous ,449 Coding-synonymous Intron 760 1, ,541 Locus-region ,976 Mrna-utr ,534 NA 3,154 4, ,183 Nonidentical Splice-site Total 4,312 6,470 1,017,246 Additionally, Deletions appear more biased away from genes than duplications (this suggests that some genes in the genome are dosage sensitive) Large deletions appear 3x smaller than large duplications (43kb versus 120kb, Redon et al., 2006) Conrad et al., 2006 Nat Genetics 19 Deletion gene biological process % deletions - % genome Biological process unclassified Nucleoside, nucleotide and nucleic acid metabolism transport Cell proliferation and differentiation Cell cycle apoptosis Amino acid metabolism Development processes Electron transport Cell structure and mobility Intracellular protein traffic Other metabolism Blood circulation and gas exchange Lipid, fatty acid and steroid metabolism Oncogenesis Coenzyme and prosthetic group metabolism Protein targeting and localization Carbohydrate metabolism Muscle contraction Protein metabolism and modification Neuronal activities Cell adhesion Immunity and defense Sensory perception Signal transduction 20 genome % deletions - % g Deletion gene molecular function Molecular function unclassified nucleic acid binding Transcription factor transporter hydrolase Signaling molecule oxidoreductase Membrane traffic protein chaperone Miscellaneous function Isomerase Lyase Extracellular matrix Ligase Select calcium binding protein Synthase and synthetase Cytoskeletal protein Transfer/carrier protein Ion channel Cell junction protein Transferase Phosphatase Select regulatory molecule Cell adhesion molecule kinase Protease Defense/immunity protein Receptor 21 7

8 Mammals share patterns of CNV polymorphism Genes from the same functional categories, and often the same genes, show tolerance to CNV across all mammalian species examined thus far (Human, Chimp, Macaque, Mouse, Rat) The data suggests that this is largely due to neutral forces, not positive/balancing selection Deletions at these loci are probably recessive (i.e no phenotype is observed in heterozygotes) GO categories significantly overrepresented in mouse CNVs Duplications probably have no adverse effect Graubert et al., (2007) PLoS Genetics 22 Dosage sensitivity Change in copy number of a dosage sensitive genes leads to an abnormal phenotype Haploinsufficient genes are those specifically sensitive to deletion Position of a gene in a gene network is a useful predictor of dosage sensitivity Safely duplicated gene Non-duplicated gene Korbel et al., (2008) Current Opinion in Structural Biology 23 Selection in Drosophila 312 CNVs/genome detected in Emerson et al. 1 every 12kb Ratio of duplications:deletions 2.5:1 Estimate fitness impact of duplications (γ < 0 => negative impact on fitness) As a class, most CNVs appear to be neutral or deleterious 24 8

9 Malaria and CNV Two common deletions confer resistance to malaria: Deletions of alpha-globin (results in alpha-thalassemia) Deletion of AE1 cause Southeast Asian Ovalocytosis In both cases, the connection with malaria was made by recognizing that the geographic distribution of the mutation was correlated with rates of endemic malaria Healthy red blood cells Ovalocytic RBCs Wrong et al., (2002) Kidney International 25 The importance of duplicated genes Classic statement of the theory due to Susumu Ohno (Evolution by Gene Duplication, 1970) A newly duplicated gene is freed from functional contraint and can head explore previously forbidden mutations, and potentially gain new functions (neofunctionalization) Homeobox gene clusters evolved by duplication; The duplication is mirrored in the segmented body of Drosophila legs only halteres and legs legs and wings Antennapedia Bithorax 26 The fate of duplicated genes Regulatory elements Transcribed region Full function Dead function New function duplication Phase I nonfunctionalization neofunctionalization subfunctionalization Phase II Force et al., (1999) Genetics 27 9

10 Taming genome complexity SMAvar1 Seg dups SMAvar1 SMAvar2 Seg dups SMAvar2 Schmutz et al., (2005) Nature 28 Conclusions Near complete ascertainment of large, common CNVs in Europeans Only true for events assayable with clone/oligo arrays Extreme heterogeneity in mutation processes and rates LD, demographic properties of CNVs very similar to SNPs CNVs are not marker of choice for demographic inference Evidence for selection on CNVs Mostly purifying selection Interesting ways in which CNVs can achieve functional changes that SNPs cannot Small number of copy number variants confer traits that are critical to our species success and survival