Genome Structural Variation in Human and Primate Evolution

1. Genome Structural Variation in Human and Primate Evolution James M. Sikela, Ph.D. Professor, University of Colorado Denver School of Medicine Genomics Course Lecture, February 22, 2011

2. Key Points • All regions of the human genome are not created equal • Gene duplication (copy number variation) is a (the?) major mechanism underlying genome evolution • ArrayCGH can reconstruct the evolutionary history of gene duplication & loss in the human/primate genome • Lineage-specific gene duplications are candidates to underlie lineage-specific traits • Selection of evolutionarily adaptive sequences also is a key driver of human disease • Human lineage-specific amplification of DUF1220 protein domains as a candidate underlying human brain evolution

3. Primate Family Tree Smithsonian Human Origins Program

4. Body shape and thorax Cranial properties (brain case and face) Small canine teeth Skull balanced upright on vertebral column Reduced hair cover Enhanced sweating Dimensions of the pelvis Elongated thumb and shortened fingers Relative limb length Neocortex expansion Enhanced language & cognition Advanced tool making Human Characteristics modified from S. Carroll, Nature, 2005

5. Reports of “human-specific” genes • FOXP2 • Mutated in family with language disability • Two human-specific amino acid changes • ASPM/MCPH • Mutated in individuals with microcephaly • Under positive selection? • HAR1F • Encodes RNA (not protein) product • Gene sequence highly changed in humans • DUF1220 protein domains • Highly increased in copy number in humans • Copy number correlation with microcephaly/macrocephaly • Expressed in important brain regions

6. Molecular Mechanisms Underlying Genome Evolution • Single nucleotide substitutions - change gene expression & structure • Genome rearrangements • Gene duplication - copy number change: gene dosage - redundancy as a facilitator of innovation

7. Strategies to identify human lineage-specific genomic changes Comparative genomic sequencing Chimp genome sequence 2005 HAR1F, 2006 Cross-species brain gene expression profiling Human, chimp, macaque Comparative genomic copy number studies: gene duplication & loss Fortna, et al, 2004: Human and great ape lineages

9. Comparative analysis of primate genome WGS sequences Genome-wide comparison of inter-species gene copy number and structural variation Evolutionary studies of disease genes associated with cognitive dysfunction e.g. MCPH Genes and genomic changes underlying human-specific cognitive capabilities Comparative brain gene expression studies Functional testing of candidate genes Sikela, J.M., PLoS Genet.2, e80, 2006

10. Gene Duplication & Evolutionary Change “There is now ample evidence that gene duplication is the most important mechanism for generating new genes and new biochemical processes that have facilitated the evolution of complex organisms from primitive ones.”- W. H. Li in Molecular Evolution, 1997 “Exceptional duplicated regions underlie exceptional biology”- Evan Eichler, Genome Research11:653-656, 2001

11. Interhominoid cDNA Array-Based Comparative Genomic Hybridization (aCGH) Fig 1. Measuring genomic DNA copy number alteration using cDNA microarrays (array CGH). Fluorescence ratios are depicted in a pseudocolor scale, such that red indicates increased, and green decreased, gene copy number in the test (right) compared to reference sample (left).

12. Experimental Design • Carry out pairwise aCGH comparisons between human and other primate species • Use a microarray containing >41,000 human cDNAs representing >24,000 human genes • Hybridize human genomic DNA (reference sequence: green) and other primate genomic DNAs (test sequence: red) simultaneously to the microarray • Visualize aCGH signals “gene-by-gene” along each chromosome across five species: human (n=5), bonobo (n=3), chimpanzee (n=4), gorilla (n=3) and orangutan (n=3)

13. Whole Genome Caryoscope Image of Interhominoid aCGH Data

15. Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss Fortna, et al, PLoS Biol. 2004

16. Clustering of hominoid lineage-specific genes

17. H C G O Value of Outgroup Comparisons: Chimp vs Human CNVs are not necessarily lineage-specific

18. aCGH Caveats • Functional status of extra copies unknown • Small copy number changes in large, highly similar gene families difficult to detect • Genes “lost” in human lineage will be missed • “Lineage-specific” term needs better validation: • More individuals needed within each species • More species need to be assayed to identify “lineage-specific” changes

19. Conclusions from Fortna, et al • First genome-wide & first gene-based survey of gene duplication & loss in human and great ape evolution • Identified most of the major lineage-specific gene copy number changes that have occurred over the past 15 million years of human and great ape evolution • Identified genes that potentially underlie many of the phenotypic characteristics that distinguish these species from one another

20. “This (Fortna, et al, 2004) is the first time that copy number changes among apes have been assayed for the vast majority of human genes, and we can expect that the biological consequences of the 140 human-specific copy number changes identified in this study will be heavily investigated over the coming years. “ ---M. Hurles, PLoSBiol. 2004

21. Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

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23. DUF1220 Repeat Unit Popesco, et al, Science 2006

24. InterPro-predicted DUF1220-containing proteins

25. Q9H094_HUMAN/236-298 Q9C0H0_HUMAN/138-201 Q8ND86_HUMAN/334-400 Q8IX77_HUMAN/116-178 100 100 100 100 80 80 # of BLAT Hits 80 80 # of BLAT Hits # of BLAT Hits # of BLAT Hits 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 MUS PTR MMU PTR RAT RAT CFA CFA PTR PTR MMU CFA CFA BTA MUS RAT BTA BTA MUS MMU BTA RAT MMU MUS HSA HSA HSA HSA Q8IX62_HUMAN/111-177 O95877_HUMAN/28-94 Q8ND86_HUMAN/184-250 Q8IX62_HUMAN/186-252 100 100 100 100 80 80 80 80 # of BLAT Hits # of BLAT Hits # of BLAT Hits # of BLAT Hits 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 RAT MUS RAT BTA RAT MUS PTR CFA MMU MUS RAT PTR BTA CFA MUS BTA BTA MMU PTR MMU CFA PTR MMU CFA HSA HSA HSA HSA C O75042_HUMAN/1586-1638 Q8IX62_HUMAN/17-83 Q8IX71_HUMAN/95-158 4 100 15 # of BLAT Hits 3 80 # of BLAT Hits 10 # of BLAT Hits 60 2 40 5 1 20 0 0 0 XTR DME BTA PTR RAT MDO CFA MMU CFA BTA MUS GGA MUS RAT CFA MUS RAT PTR MMU BTA PTR MMU HSA HSA HSA BLAT Estimation of the Number of DUF1220 Domains Found in Different Species A B

26. Orangutan Macaque Bonobo Baboon Gibbon Human Gorilla Chimp Copy Number of DUF1220 (Q8IX62/17-33) Sequences in Primate Species 70 60 50 Q-PCR Predicted Copy Number 40 30 20 10 0

27. BLAT-based DUF1220 copy number in sequenced primates using IMAGE:843276 • Full insert cDNA query (491 bp) encodes 3 DUF1220 domains • BLAT hits (>200 score) in each species: • Species (Assembly) Copies (x3) • Human (5/04): 51 • Human (3/06): 50 • Chimp (11/03): 6 • Chimp (3/06): 25 • Orangutan (7/07): 10 • Macaque (1/06): 3

28. Summary of aCGH, Q-PCR and BLAT results: • DUF1220 domains are highly amplified in human, reduced in African great apes, further reduced in orangutan and Old World monkeys, single copy in non-primate mammals and absent in non-mammals

29. Sequences encoding DUF1220 domains: • are virtually all primate specific • are increasingly amplified generally as a function of a species evolutionary proximity to humans, where the greatest number of copies (218) is found • show signs of positive selection • are highly expressed in brain regions associated with higher cognitive function • in brain show neuron-specific expression preferentially in cell bodies and dendrites Popesco, et al, Science 2006

30. Recent Relevant Publications • Fortna, A., Kim, Y., MacLaren, E., Marshall, K., Hahn, G., Meltesen, L., Brenton, M., Hink, R., Burgers, S., Hernandez-Boussard, T., Karimpour-Fard, A., Glueck, D., McGavran, L., Berry, R., Pollack, J.R. and Sikela, J.M.: Lineage-specific gene duplication and loss in human and great ape evolution. PLoS Biology, Jul;2(7):E207, 2004. • Sikela, J.M.: The Jewels of Our Genome: The Search for the Genomic Changes Underlying the Evolutionarily Unique Capacities of the Human Brain. PLoS Genet, May;2(5):e80, 2006. • Popesco, M., MacLaren, E., Hopkins, J., Dumas, L., Cox, M., Meltesen, L., McGavrin, L, Wyckoff, G., and Sikela, J.M.: Human lineage-specific amplification, selection and neuronal expression of DUF1220 domains. Science, 313:1304-1307, 2006. • Dumas, L., Kim, Y., Karimpour-Fard, A., Cox, M., Hopkins, J., Pollack, J., and Sikela, J.M.: Gene copy number variation spanning 60 million years of human and primate evolution. Genome Research 17:1266-1277, 2007. • Dumas L. and Sikela, J.M.: DUF1220 Domains, Cognitive Disease and Human Brain Evolution. Cold Spring Harb. Symp. Quant. Biol. E-published, October 22, 2009.