copy number variations: a new type of genetic marker in whole-genome association studies

1. Copy Number Variations: a new type of genetic marker in whole-genome association studies Wentian Li, Ph.D Center for Genomics and Human Genetics Feinstein Institute for Medical Research North Shore LIJ Health System Oct 31, 2008 Consumer Genomics Workshop Center for Genetic Medicine Northwestern University

3. terminology Copy number variation ( germline, inherited) = inherited: also present in parents� genome = de novo: absent in parents� genome Copy number alteration (somatic, e.g. in cancer cells) Copy number polymorphism (relatively common CNV, with a fixed starting/ending position)

4. Overview of structural aberration Different length scales CGH/arrayCGH/ROMA 2. Using genotyping array for CNV study 3. Examples of disease mapping 4. Practical issues

5. length scales of aberrations/variations/polymorphisms

6. �structural aberration� (1) whole genome duplication Polyploidy is common in plants (Rare in animals) Survival rate after WGD may be very low. Major genomic instability would follow including massive gene losses In vertebrates, WGD is thought to occur twice around 500 million years ago (2R hypothesis)

7. Structural variation (2)gain or loss of certain chromosomes Aneuploidy: monosomy[1], trisomy[3], tetrasomy[4] either fatal (spontaneous abortion) or responsible for abnormal phenotypes Chromosome-specific aneuploidy rate? less number of chiasmata -- shorter chromosomes: ch21,ch22 Down syndrome: trisomy 21

9. Structural aberration (3):microscopically-visible aberrations Breaks Double-breaks (inversion, translocation) Deletions (4p, 5p, 9p, 11p/11q, 13q, 18p/18q). deletion syndromes Duplications (inverted 15p). Iso-chromosomes are inverted duplications of the whole arm. �balanced� vs. �unbalanced� (deletion/loss, duplication/gain)

10. translocation between ch7 and ch13: �balanced�

11. extra copy on ch16, extra two copies on ch6: �unbalanced�

12. Chromosome CGH: comparative genome hybridization (Pinkel 1992) advantages: No need to prepare chromosomes, only DNA Simple color scheme: e.g.,duplications show up as red, deletions as green, normal as yellow disadvantages: Need sophisticated microscopic/image analysis Long time (~days) in hybridization Time-consuming analysis of the result

13. Structural aberration (4): from microscopic to submicroscopic, from chromosome CGH to array CGH (Pinkel/Albertson 2001)

14. ROMA: representational oligo-nucleotide microarray analysis (Lucito/Wigler 2003)

15. Overview of structural aberration Using genotyping array for CNV study R-ratio and theta series Cumulative plots Hidden Markov model 3. Examples of disease mapping 4. Practical issues

17. The basic idea behind CNV detection using genotyping array

18. Hemizygous deletion (CN=1)

19. Homozygous deletion (CN=0)

20. Duplication (CN=3)

21. Delineate CNV regions Eyeballing the theta and R-ratio plots (for large CNV regions) Cumulative plots Hidden Markov model

22. CNA in cancer cell: chronic lymphocytic leukemia (black: normal, blue: cancer cell) [ch13]

23. cumulative plot, detrended cp

24. Combining two cumulative plots into one � for hemizygous deletion

25. � for homozygous deletion

26. CLL, ch6

27. Hidden Markov models

28. Some HMM-based CNV detection programs QuantiCNV www.well.ox.ac.uk/QuantiSNP/ PennCNV www.neurogenome.org/cnv/penncnv/ dChip biosun1.harvard.edu/complab/dchip/copy.htm

29. advantages and points-to-consider (HMM) Using information from R-ratio and theta series simultaneously Standard algorithm Using the same set of parameter throughout the sequence implies that heterogeneity is not allowed The fixed parameter implies a characteristic length for CNV regions

30. Overview of structural aberration Using genotyping array for CNV study Examples of disease mapping Autism Schizophrenia Crohn�s disease 4. Practical issues

32. Example(1): Autism Brain development disorder Age of diagnosis: 3 Impairment in social interaction, in communication, restricted interests, repetitive behavior �autism spectrum disorder�: Pervasive Developmental Disorder - Not Otherwise Specified Concordance rate in MZ twin: 70%/90%, in DZ twin: 5%/10%

33. Sebat et al. Science (Apr 20, 2007)

34. Example (1): More details Roughly 200 patients and 200 controls (patients either have, or do not have, affected siblings) ROMA technology is used: resolution is 35kb 14 CNVs detected in 195 ASD, 2 CNVs in 196 controls (statistically significant) Out of 14 CNVs, 12 in sporadic cases, 2 in multiplex families 12 out of 15 CNVs in cases are deletions, the 2 CNVs controls are duplications

36. Example(2): Schizophrenia Mental disorder Auditory hallucinations, paranoid delusions, disorganized speech and thinking. Age of onset: early adulthood Concordance rate for MZ twins: 48%, for DZ twins: 4%

38. ISC paper: more details ~3000 cases and ~3000 controls Affymetrix Human SNP 5.0 and 6.0 array 6751 (>100kb) CNVs are detected. 1.14 CNV per person in cases, 0.99 in controls Various attempts to increase the odd-ratio (gene load, single CNV, larger-sized CNV), but more or less the same Confirming a known risk deletion on ch22q11: 13 in cases, none in controls Other 271 (>500kb) deletions (161 in cases,110 in controls). 15q13 (new). 1q21.

40. deCODE paper: more details 1433 cases, 33250 controls, followed by 3285 cases, 7951 controls Illumina HumanHap300, 550, Affymetrix GenomeWide 6.0 Only search for de novo CNV (in 9878 parent-child transmissions): 66 are found Three deletions: 1q21.1, 15q11.2, 15q13.3

42. Example(3): Crohn�s disease(McCarroll et al. Nat.Genet.2008)

44. Contributions of these new CNV studies Autism: new explanation on why the concordance rate in MZ twins is high: the twins share the same deletion/duplication event. Schizophrenia: narrowing the 22q11.2 risk deletion region from 17-21Mb to 3Mb Crohn�s disease: within the associated region/gene, locate the causal mutation

46. Overview of structural variations Using genotyping array for CNV study Examples of disease mapping Practical issues common CNPs vs. de novo CNVs

47. Common vs. de novo CNV Present in general population with a fixed starting/ending positions Similar to SNP, especially SNPs with the same frequency Are they already captured by SNPs?

48. Common CNV

49. Common vs. de novo CNV More deleterious. The evolutionary negative selection makes it more relevant to diseases like autism and schizophrenia. de novo CNVs should be easier to detect than de novo SNPs (until we have cheaper sequencing technologies) Similar to cytogenetic studies (either genetic or cancer studies): smaller sample sizes, individually distinct mutations

50. de novo CNV

51. Share similar issues as SNP-based whole-genome association studies ethnic/population stratifications missing-typing rates differ between case and control groups (usually the hetero-zygotes are most likely to be untyped) Multiple testings

52. summary CNV as a new detectable source of variation/mutation/polymorphism should not be overlooked Studies on de novo CNV in autism and schizophrenia represent a new promising strategy CNV hot spots (e.g. segmental duplication regions) Relevance of common CNVs to common diseases is to be examined

53. Source materials Feuk et al (2006), �Structural variations in the human genome�, Nat. Rev. Genet.7:85-97. URL: www.nslij-genetics.org/duplication/ (600 papers) Peiffer, Gunderson (2006), �SNP-CGH technologies for genomic profiling of LOH and copy number�, Clinical Laboratory International (May06). Li, Lee, Gregersen (2009), �Copy-number-variation and copy-number-alteration region detection by cumulative plots�, BMC Bioinf, to appear. Sebat et al (2007), �Strong association of de novo copy number mutations with autism�, Science, 317:445-449. Stefansson et al. (2008), �Large recurrent microdeletions associated with schizophrenia�, Nature, 455:232-236. Int. Schizophrenia Consortium (2008),�Rare chromosomal deletions and duplications increase risk of Schizophrenia�, Nature, 455:237-241. McCarroll et al. (2008), �Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease�, Nature Genet. 40:1107-1112. McCarroll (2008), �Extending genome-wide association studies to copy-number variation�, Hum. Mol. Genet. 17:R135-R142 URL: www.nslij-genetics.org/cnv/ (300 papers)