Structural Variants and Susceptibility to Common Human Disorders Dr. Xavier Estivill

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1 Structural Variants and Susceptibility Genetic Causes of Disease Lab Genes and Disease Program Center for Genomic Regulation (CRG) Barcelona 1 Complex genetic diseases Changes in prevalence (>10 fold) 2 Genome variability is behind the biological differences in health and disease 3 1

Genetic basis of complex genetic diseases Common disease common variant

effect (Rare variants with frequencies <0.

variants can cause disease) 4 Genome wide association studies 2007-2008

Several successful GWA studies performed Robust and replicated

Modest genetics effects with odd ratios <1.

2 Genetic basis of complex genetic diseases Common disease common variant (No more than 5% of susceptibility to disease) Rare alleles of major effect (Rare variants with frequencies <0.01 can cause disease) Infinitesimal model (Hundreds or thousands of variants can cause disease) 4 Genome wide association studies Successful identification of genetics associations for common disorders Several successful GWA studies performed Robust and replicated associations detected Proof of concept that SNP genotyping works! Modest genetics effects with odd ratios <1.5 Finding do not explain the strong heritability Variants are not the functional ones Absence of epistasis between variants 5 Human genome variation 6 2

1980 1990 2002 (1975-1980) Description of copy number variation of the α-globin genes by Kan and co-workers

type 1A Identification that a large subset of SNPs are paralogous sequence variants (PSVs) and define regions of

demonstrated the existence of copy number variants (CNVs) Redon et al.

of structural variants Duplication nsequences 20,000 CNVs >6,000 CNV loci Del 7 Chr7 Test Control Functional co

3 ( ) Description of copy number variation of the α-globin genes by Kan and co-workers Genomic rearrangements are identified as the mutational mechanism that leads to Charcot- Marie-Tooth disease type 1A Identification that a large subset of SNPs are paralogous sequence variants (PSVs) and define regions of structural variability Interrogation of genomic variability by array hybridization methods demonstrated the existence of copy number variants (CNVs) Redon et al. identified 1,447 copy number variable regions showing that at least 12% of the human genome contains CNVs 7 Type of structural variants Duplication nsequences 20,000 CNVs >6,000 CNV loci Del 7 Chr7 Test Control Functional co Structural variations involve between 5% and 10% of the human genome sequence Estivill and Armengol, PloS Genetics, October copies 12 cop es Common Rare invers on 8 9 3

Detection of CNVs by WGTP and SNP arrays Comparative genome

Test Reference Test DNA DNA DNA DNA Comparative intensity

150 Mb Log 2 (test/reference) Redon et al.

Structural Variation Consortium (Nature 2006) 10 CNV algorithms

Number of consecutive SNPs PennCNV: Reference population Number

4 Detection of CNVs by WGTP and SNP arrays Comparative genome hybridisation: Whole genome TilePath (WGTP array) Reference Test Reference Test DNA DNA DNA DNA Comparative intensity analysis: Affymetrix 500K access SNP chip DNA 1 DNA 1 DNA 2 DNA 2 NspI StyI NspI StyI Genome profile Combine dye-swaps Combine chips Compare samples Combine chips Log 2 (test/reference) Chromosome profile Chromosome 8 Chromosome 8 Log 2 (test/reference) 10 Mb window 50 Mb 100 Mb 150 Mb 50 Mb 100 Mb 150 Mb Log 2 (test/reference) Redon et al Mb 4 Mb 6 Mb 8 Mb 10 Mb 2 Mb 4 Mb 6 Mb 8 Mb 10 Mb Genome Structural Variation Consortium (Nature 2006) 10 CNV algorithms comparison Variable parameters: GADA: False discovery rate (T) Number of consecutive SNPs PennCNV: Reference population Number of consecutive SNPs Size of variation QuantiSNP: Log Bayer factor 11 CNV analysis and algorithm testing 12 4

CNV algorithms comparison SNPs from different Illumina platforms located within CNVs Distinct probe count Image from Cooper et

5 kb 1,500 CNV with at least two variants in 270 HapMap samples >350 common CNVs (allele frequency >5%) in Europeans (CEU) 42 M

, WTSI, unpublished) >50,000 CNV calls made using GADA algorithm ~ 12,000 CNVs identified ~ 1,300 CNVs between two individuals

5 CNV algorithms comparison SNPs from different Illumina platforms located within CNVs Distinct probe count Image from Cooper et al., New generation CNV studies Affymetrix 6.0 SNP array (McCarroll et al., 2008, Nat. Genet.) Median length of 7.5 kb 1,500 CNV with at least two variants in 270 HapMap samples >350 common CNVs (allele frequency >5%) in Europeans (CEU) 42 M Nimblegen oligonucleotide acgh (Hurles et al., WTSI, unpublished) >50,000 CNV calls made using GADA algorithm ~ 12,000 CNVs identified ~ 1,300 CNVs between two individuals ~ 1% of the genome varies in CNV between two diploid genomes 14 Next generation sequencing Capacity greater than one Gigabase per run Drastic decrease in costs per genome Applications: DNA, RNA, Chromatin etc. 15 5

6 Pair End Mapping Tuzun et al., Nature Genetics, 2005 Korbe et al., Science 2007 Kidd et al., Nature GSTM CNV and breakpoints GSTT CNV and breakpoints 17 Polymorphic inversions in humans The combination of the predictions of different studies has resulted in 348 independent inversions in humans, although the overlap is low: Levy et al., 2007 (90 indep. inv.) Korbel et al., 2007 (88 indep. inv.) Kidd et al., 2008 (245 indep. inv.) Alexander Martinez / Mario Caceres 18 6

Effects of rare CNVs on complex disorders Early-onset Parkinson s Disease (SNCA) Hereditary early-onset Alzheimer s Disease (APP) Hereditary

complex disorders HIV-1/AIDS susceptibility (CCL3L1) Microscopic Polyangiitis (FCGR3) Wegener s Granulomatosis (FCGR3) Systemic Lupus Erythematosus

rejection (CCL4L) Osteoporosis (UGT2B17) Body Mass Index (NEGR1) Psoriasis (LCE3C/LCE3B) 20 FCGR3 and FCGR2 CNV and autoimmune disorders (Microscopic

, 2008 cen tel Mb 159.74 159.79 159.84 159.89 159.95 159.

7 Effects of rare CNVs on complex disorders Early-onset Parkinson s Disease (SNCA) Hereditary early-onset Alzheimer s Disease (APP) Hereditary Pancreatitis (PRSS1) Autism Spectrum Disorders (several genes) Cancer (several genes) Schizophrenia (several genes) 19 Effects of common CNVs on complex disorders HIV-1/AIDS susceptibility (CCL3L1) Microscopic Polyangiitis (FCGR3) Wegener s Granulomatosis (FCGR3) Systemic Lupus Erythematosus (FCGR3 and C4A/C4B) Crohn s Disease (DEFB4 and IRGM) Idiopathic Thrombocytopenic Purpura (FCGR2C) Rheumatoid Arthritis (CCL3L1) Lung transplantation rejection (CCL4L) Osteoporosis (UGT2B17) Body Mass Index (NEGR1) Psoriasis (LCE3C/LCE3B) 20 FCGR3 and FCGR2 CNV and autoimmune disorders (Microscopic Polyangiitis, Wegener s Granulomatosis, Lupus, Idiopathic Thrombocytopenic Purpura) Aitman et al., 2006 Fanculli et al., 2007 Breunis et al., 2008 cen tel Mb Genes FCGR2A FCGR3A FCGR2C FCGR3B FCGR2B FCGRLA FCGRLB Segmental Duplications Copy number variants Affymetrix GAP Illumina GAP Linkage disequilibrium blocks HSPA6 82 Kb 82 Kb Loss (chr1.85) 98% Sebat et al Loss Sebat et al Gain and Loss (CNV62) Redon et al

Gene copy-number variation and associated polymorphisms of complement component C4 in human system lupus erythematosus Yang et al.

11 Genes STK19 TNXB C4A C4B CYP21A2 Segmental Duplications 33 kb 99% 33 kb Copy number variants Affymetrix GAP Illumina GAP HapMap Gain

2005 22 Common copy number variants in common disorders Fibromyalgia Multiple Sclerosis Psoriasis Asthma Migraine Stroke Fibromyalgia

Rabionet, Eva Riveira 23 Psoriasis: a chronic skin disorder with increasing prevalence Common chronic inflammatory disease

8 Gene copy-number variation and associated polymorphisms of complement component C4 in human system lupus erythematosus Yang et al., 2007 tel cen Chromosome 6p21 Mb Genes STK19 TNXB C4A C4B CYP21A2 Segmental Duplications 33 kb 99% 33 kb Copy number variants Affymetrix GAP Illumina GAP HapMap Gain and Loss (Chr6.3) Tuzun et al Gain and Loss (CNV504) Redon et al Gain and Loss (Chr6.2) Tuzun et al Common copy number variants in common disorders Fibromyalgia Multiple Sclerosis Psoriasis Asthma Migraine Stroke Fibromyalgia Multiple Sclerosis Psoriasis Asthma Migraine Stroke Luis Armengol, Rafa de Cid, Anna Brunet, Elisa Docampo, Eulalia Marti, Kelly Rabionet, Eva Riveira 23 Psoriasis: a chronic skin disorder with increasing prevalence Common chronic inflammatory disease White-silvery scaly plaques Abnormal keratinocyte proliferation Activation of innate immunity pathways Br J Dermatol 152: , 2005 Worldwide prevalence variation Commonest in Scandinavia and Northern Europe: 3% North America and the UK: 2% Japan: 0.2% 24 8

9 Multifactorial aetiology of Psoriasis 25 Susceptibility loci for Psoriasis Strongest association with HLA class I alleles on Chr 6p21 PSORS1: HLA-Cw* %-50% of familial clustering Non-HLA loci: PSORS2 on Chr 17q25 PSORS6 on Chr 19p13 PSORS3 on Chr 4q35 PSORS7 on Chr 1p35-34 PSORS4 on Chr 1q21 PSORS8 on Chr 16q12-13 PSORS5 on Chr 3q21 PSORS9 on Chr 4q31-34 IL12B IL23R DEFB4 26 Identification of CNVs determinants in Psoriasis 27 9

Dosage genomic changes identifies in Psoriasis Criteria for copy number change Entry probe consecutive probes Log 2 value >0.3 or <-0.3 >0.25 or <-0.25 Same direction 28 Chr 1q21.

A_16_P15296703 A_14_P112866 A_14_P100633 A_16_P00165273 CNVs LCE3C LCE3B del 29 Replication in four independent sample sets 3 population-based Population Phenotype Alleles Spanish (557) Italian (900)

(55%) 573 (64%) 516 (57%) 281 (69%) 334 60 847 (71%) 378 (64%) 1926 (68%) 1645 (59%) LCE3C LCE3B 125 (36%) 344 (45%) 327 (36%) 384 (43%) 127 (31%) 226 40 345 (29%) 210 (36%) 924 (32%) 1164 (41%) OR 1.

10 Dosage genomic changes identifies in Psoriasis Criteria for copy number change Entry probe consecutive probes Log 2 value >0.3 or <-0.3 >0.25 or <-0.25 Same direction 28 Chr 1q ,800 k 150,900 k Genes LCE5A CRCT1 LCE3E LCE3D LCE3C LCE3A LCE3B 150,830 k 150,840 k 150,850 k LCE2D LCE2B LCE2C LCE2A Late cornified envelope Agilent 244k A_16_P A_16_P A_16_P A_14_P A_14_P A_16_P CNVs LCE3C LCE3B del 29 Replication in four independent sample sets 3 population-based Population Phenotype Alleles Spanish (557) Italian (900) Dutch 484 US (890) Total (2831) Psoriasis Control Psoriasis Control Psoriasis Control Psoriasis Control Psoriasis Control LCE3C LCE3B-del 225 (64%) 420 (55%) 573 (64%) 516 (57%) 281 (69%) (71%) 378 (64%) 1926 (68%) 1645 (59%) LCE3C LCE3B 125 (36%) 344 (45%) 327 (36%) 384 (43%) 127 (31%) (29%) 210 (36%) 924 (32%) 1164 (41%) OR 1.47 ( ) 1.30 ( ) 1.50 ( ) 1.36 ( ) 1.38 ( ) ( ) ( %) ( %) ( ) P-value E 08 1 family-based Population Informative families Alleles Founders NT allele P-value US 308 LCE3C_LCE3B non_del 0.65/ / E

11 Linkage disequilibrium across the LCE cluster HapMap SNPs Genotyped SNPs Haploblocks 31 SNPs spanning >100 are in LD with the LCE3C-LCE3B deletion 32 rs is a proxy of the LCE3C-LCE3B deletion Chr1q ,800 k 150,900 k Genes LCE5A CRCT1 LCE3E 32 kb del LCE3C LCE3D LCE3B LCE3A LCE2D LCE2B LCE2C LCE2A PopulationPsoriasis Control rs p 2.4 E 126 rs is associated to Psoriasis Dominant (CC CT) vs. TT Recessive CC vs. (CT TT) Log additive OR 95%CL OR 95%CL OR 95%CL P-value r 2 Spanish ( ) 2.70 ( ) 1.61 ( ) 4.9E

12 Association of rs genotypes in the LCE cluster with Psoriasis in Spanish, Italian, Dutch and US samples Overall OR are standardized by population according to a logistic model in which population was introduced as a confounding variable after (nonsignificant) heterogeneity was detected by population 34 Genetic interaction analysis between LCE3C_LCE3B-del and LCE3C_LCE3B-del tag SNP and PSORS1 loci 35 Genetic interaction analysis between LCE3C_LCE3B-del and LCE3C_LCE3B-del tag SNP and PSORS1 loci (2) Haplotypes of the PSORS1 locus associated to Psoriasis in Spanish samples 36 12

13 Interaction analysis between LCE and HLA genes OR = Genetic interaction analysis between LCE3C_LCE3B-del and LCE3C_LCE3B-del tag SNP and PSORS1 loci (3) 38 Chromosome 1q ,800 k 150,900 k LCE5A CRCT1 LCE3E LCE3D LCE3C LCE3B LCE3A LCE2D LCE2B LCE2C LCE2A 150,830 k 150,840 k 150,850 k LCE3C LCE3B LCE3C LCE3B-del What are the consequences of the LCE3B_LCE3C deletion at the expression level? 39 13

14 Expression levels of LCE3C mrna in epidermal cells depend on LCE3C copy number 40 Patterns of expression of LCE3C in the epidermis LCE genes are expressed at low to undetectable levels in normal skin, but are strongly expressed in individuals with Psoriasis 41 Patterns of expression of LCE3C in the epidermis (2) Only lesional epidermis of Psoriasis patients showed LCE3C expression 42 14

Patterns of expression of LCE3C in the epidermis (3) Injured skin shows a high level of expression of LCE3C and LCE3E 43

profiles of neighbour genes of the LCE cluster 44 Copy number variants in Psoriasis In a genome-wide CNV scan we have found

is significantly associated (p=1.15e08) with risk of Psoriasis (OR=1.

effects with HLA locus LCE3C and LCE3B are the first non-immunity related genes involved in Psoriasis susceptibility LCE3C

15 Patterns of expression of LCE3C in the epidermis (3) Injured skin shows a high level of expression of LCE3C and LCE3E 43 Expression levels of LCE genes with respect to LCE SNPs LCE3C and LCE3B absence could have effects on the expression profiles of neighbour genes of the LCE cluster 44 Copy number variants in Psoriasis In a genome-wide CNV scan we have found strong statistical evidence of association between deletion of LCE3C and LCE3B and Psoriasis The absence of LCE3C and LCE3B is significantly associated (p=1.15e08) with risk of Psoriasis (OR=1.38) in several populations rs is a tag SNP of the LCE3C_ LCE3B deletion LCE3C_LCE3B deletion shows multiplicative effects with HLA locus LCE3C and LCE3B are the first non-immunity related genes involved in Psoriasis susceptibility LCE3C expression is induced upon skin injury and in Psoriasis Loss of LCE3C / LCE3B or altered expression of LCE genes might lead to compromised skin barrier function and Psoriasis susceptibility 45 15

16 New concept in Psoriasis pathophysiology: compromised skin barrier function and Psoriasis susceptibility 46 Chromosomal plot (1,139 cases + 1,132 controls) LCE3A/D MHC (p 1,93E- 208) logp observed -log 10 (P) IL12B logp expected Chromosomal location

Global variation in copy number in the human genome

Global variation in copy number in the human genome Redon et. al. Nature 444:444-454 (2006) 12.03.2007 Tarmo Puurand Study 270 individuals (HapMap collection) Affymetrix 500K Whole Genome TilePath (WGTP)