0.1% variance attributed to scattered single base-pair changes SNPs

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1 April 2003, human genome project completed: 99.9% of genome identical in all humans 0.1% variance attributed to scattered single base-pair changes SNPs

2 It has been long recognized that variation in the human genome exists and can range from single nucleotide changes to large chromosome anomalies. These known variations have been considered to give rise to the genetic or familial component of many common complex diseases, including autoimmune diseases. However, recent large-scale genome-wide association studies of seven common diseases, including autoimmune diseases, using single nucleotide polymorphism (SNP) analysis concluded that genetic "association signals so far identified account for only a small proportion of overall familiality" and "These estimates demonstrate the limited potential of the variants thus far identified (singly or in combination) to provide clinically useful prediction of disease".

3 Is there another genetic variant that contributes to the familial or genetic component of common complex diseases? Down syndrome: trisomy, chromosome 21 Amplification of DHFR gene leading to methotrexate resistance in cancer Amplification of c-myc and N-Myc genes in cancer

4 Copy Number Variants Involved in Human Disease DISEASE GENE PHENOTYPE Charcot-Marie-Tooth type 1A PMP22 Demyelination, peripheral neuropathy X-linked hypopituitarism SOX3 In males, short stature, mild mental retardation Autosomal dominant leukodystrophy LMNB1 Demyelination, white brain matter abnormalities Parkinson's SNCA Neuron degeneration, rigidity, tremor Alzheimer's APP Amyloid precursor protein buildup Altered drug metabolism CYP2D6 Increased side effects, increased or decreased efficacy HIV/AIDS CCL3L1 Increased susceptibility to infection and disease Lupus FCGR3B Increased susceptibility to kidney failure Smith-Magenis syndrome RAI1 Mental retardation Pelizaeus-Merzbacher PLP1 Demyelination, paralysis of legs, involuntary jerking of head Spinal muscular atrophy SMN1 Spinal deterioration, milder disease w/later onset Rett-like syndrome MECP2 Mental retardation, spasticity, language/speech problems Miller-Dieker syndrome LIS1 Brain malformation, mental retardation, epilepsy Neurofibromatosis type 1 NF1 Tumors, cognitive deficits CREDITS (TOP TO BOTTOM): CORBIS; AP; ALIX/PHOTO RESEARCHERS; SOURCE: LEE AND J. LUPSKI/BAYLOR COLLEGE OF MEDICINE; CONRAD AND ANTONARAKIS/UNIVERSITY OF GENEVA MEDICAL SCHOOL Scherer, Lee, and other colleagues have since created a Database of Genomic Variants to catalog all reported CNVs in normal humans. As of their most recent update in March, they had compiled 6482 CNVs from 40 publications. Just this week, in the 4 September issue of

5 GENOMICS:DNA Duplications and Deletions Help Determine Health Jon Cohen Each human's genome is distinguished by extra, and sometimes missing, DNA that can powerfully impact everything from development to disease Fingering the culprit. Extra copies of one gene cause a form of Charcot-Marie-Tooth disease, a neuropathy that affects the feet and hands.credit: CHARCOT-MARIE- TOOTH ASSOCIATION

6 Numerous possibilities. The number of extra, missing, and mutated (red) PMP22 genes (PERIPHERAL MYELIN PROTEIN 22) leads to neuropathies (CMT1A and HNPP) of varying severity. HNPP = Hereditary Neuropathy with liability to Pressure Palsies. SOURCE: LEE ET AL., NEURON 52, (2006)

7 Article Nature 444, (23 November 2006) doi: /nature05329; Received 13 June 2006; Accepted 10 October 2006 Global variation in copy number in the human genome Richard Redon1, Shumpei Ishikawa2,3, Karen R. Fitch4, Lars Feuk5,6, George H. Perry7, T. Daniel Andrews1, Heike Fiegler1, Michael H. Shapero4, Andrew R. Carson5,6, Wenwei Chen4, Eun Kyung Cho7, Stephanie Dallaire7, Jennifer L. Freeman7, Juan R. González8, Mònica Gratacòs8, Jing Huang4, Dimitrios Kalaitzopoulos1, Daisuke Komura3, Jeffrey R. MacDonald5, Christian R. Marshall5,6, Rui Mei4, Lyndal Montgomery1, Kunihiro Nishimura2, Kohji Okamura5,6, Fan Shen4, Martin J. Somerville9, Joelle Tchinda7, Armand Valsesia1, Cara Woodwark1, Fengtang Yang1, Junjun Zhang5, Tatiana Zerjal1, Jane Zhang4, Lluis Armengol8, Donald F. Conrad10, Xavier Estivill8,11, Chris Tyler-Smith1, Nigel P. Carter1, Hiroyuki Aburatani2,12, Charles Lee7,13, Keith W. Jones4, Stephen W. Scherer5,6 and Matthew E. Hurles1

8 The experimental procedures for comparative genome hybridization on the WGTP array and comparative intensity analysis on the 500K EA platform are shown schematically (see Supplementary Methods for details), for a comparison of two male genomes (NA10851 and NA19007). The genome profile shows the log2 ratio of copy number in these two genomes chromosome-by-chromosome. The 500K EA data are smoothed over a five-probe window. Below the genome profiles are expanded plots of chromosome 8, and a 10-Mb window containing a large duplication in NA19007 identified on both platforms (indicated by the red bracket).

9 Affymetrix 6.0 SNP Array 1.8 million markers >906,600 SNPs >946,000 probes for detection of CNVs Evenly distributed along genome Set of probes that are variant indicate a copy number variation region (CNVR)

10 The chromosomal locations of 1,447 CNVRs are indicated by lines to either side of ideograms. Green lines denote CNVRs associated with segmental duplications; blue lines denote CNVRs not associated with segmental duplications. The length of right-hand side lines represents the size of each CNVR. The length of left-hand side lines indicates the frequency that a CNVR is detected (minor call frequency among 270 HapMap samples). When both platforms identify a CNVR, the maximum call frequency of the two is shown. For clarity, the dynamic range of length and frequency are log transformed (see scale bars). All data can be viewed at the Database of Genomic Variants (

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12 Overlapping CNVs called in five individuals are shown schematically for four loci (in blue); dashed lines indicate overlap. Copy number variable regions (CNVRs) represent the union of overlapping CNVs (in green). Independent juxtaposed CNVs (in black) are identified by requiring that only individual-specific CNVs that overlap by more than a threshold proportion be merged. Intervals encompassing CNV breakpoints (in red) are defined using platform-dependent criteria (Supplementary Methods), and contain a significant paucity of recombination hotspots76, 77 (Supplementary Table 13), which results from the enrichment of segmental duplications within which fewer inferred recombination hotspots reside.

13 Two different deletions within the SIRPB1 gene (exons, red) provide evidence for an independently recurrent deletion event. Both structural variants are probably mediated by non-allelic homologous recombination between segmental duplications (blue bars, arching lines) in direct orientation. Deletion alleles from four different individuals (G248 and ABC8 10) are depicted; deletion 2 (del2, minimal region chromosome 20: ) eliminates exon 2, whereas deletion 1 (del1, minimal region chromosome 20: ) does not. Repeat content and orientation are depicted as coloured arrows (green, long interspersed transposable element; purple, short interspersed transposable element; orange, transposon). Predicted and annotated segmental duplications are depicted as indicated.

14 Copy number variation (CNV) Amplifications and deletions in the genome - 1kb-1mb variants covering ~15% genome - <20% overlap with identified SNPs % correlation with expression data (Science 2007; 315:848-53) Causes - Nonallelic homologous recombination - Cell division (MTX & DHFR) Annual Review of Genomics and Human Genetics Vol. 3:

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16 CNV can influence gene expression directly by providing loss or gain of either the gene or a regulatory element. CNV can also influence gene expression indirectly through -Position effects -Predispose to deleterious genetic changes -Provide substrates for chromosomal change in evolution

17 a, The distribution of WGTP log2 ratios at five CNVs with genotype information. Each histogram of log2 ratios in 270 HapMap individuals exhibits three clusters, each corresponding to a genotype of a biallelic CNV, with the two alleles depicted by broken and complete bars, representing lower and higher copy number alleles, respectively. Red lines above each histogram denote log2 ratios in the 12 individuals represented in b. b, Mendelian inheritance of five CNVs in four parent offspring trios. The individual CNVs were genotyped from WGTP clones: green, Chr8tp-17E9; yellow, Chr1tp-31C8; blue, Chr5tp- 22E4; red, Chr6tp-5C12; black, Chr6tp-11A11.

18 Population clustering based upon CNV distributions: A triangle plot showing the clustering of 210 unrelated HapMap individuals assuming three ancestral populations (k = 3). The proximity of an individual to each apex of the triangle indicates the proportion of that genome that is estimated to have ancestry in each of the three inferred ancestral populations. The clustering together of most individuals from the same population near a common apex indicates the clear discrimination between populations obtained through this analysis. The clustering was qualitatively similar to that obtained previously with a similar number of biallelic Alu insertion polymorphisms on different African, European and Asian population samples60.

19 Nature Reviews Genetics 8, (August 2007) Do additional genetic variants exist that contribute to risk of developing common complex diseases such as rheumatoid arthritis?