A Genome-wide association study of Copy number variation in
schizophrenia
Andrés Ingason
CNS Division, deCODE Genetics.
Research Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen University Hospital.
Common variant – common disease
• The development of SNP microarrays providing genotypes for hundreds of thousands of markers at an affordable price has paved the way for the identification of at-risk genes for many common diseases
Nicotine addiction / lung cancer
Schizophrenia– all quiet on the western front
• Many studies using large samples have been conducted in schizophrenia (and other psyciatric disorders for that matter) since the introduction of genome-wide SNP microarrays, without producing unanimous positive results.
Why are psychatric diseases different?Why do we not find susceptibility variants?
• Reduced fecundity, associated with disorders such as autism, schizophrenia and mental retardation, places negative selection pressure on haplotypes carrying risk alleles.
• The genetic risk of diseases under such pressure is less likely to be accounted for by common low penetrant variants.
• Instead rare, more penetrant mutations at many sites across the genome may be involved and account for a proportion of the genetic risk.
Can we find rare high risk variants with current technology?
• In contrast to rare single nucleotide mutations, rare copy number variations (CNVs) can be detected using genome-wide arrays.
• This has led to the identification of CNVs associated with mental retardation and autism.
• Walsh et al. report very recently of CNVs being involved in schizophrenia as well
How can the prevalence of schizophrenia be reconciled with the
existence of rare high risk CNVs under negative selection ?
• Genomic regions flanked by low copy repeats (LCR) prone to non-allelic homologous recombination (NAHR) provide sites for recurrent CNV events
• These CNVs should appear more often as de novo upon inspection when under negative selection pressure.
Study designIdentification of de novo CNVsDosageMiner, software that identifies CNVs from chip data~2200 trios genotyped on Illumina chips~5700 parent-offspring pairs genotyped on Illumina chips~10100 transmissions to identify de novo deletions~4400 transmissions to identify de novo duplicationsQuality Control
Association Study (SGENE sample)~1,500 Schizophrenia patients genotyped on Illumina chips~30,000 controls genotyped on Illumina microarrays
DosageMiner
• Signal intensities from SNPs on Illumina chips are normalized to reduce noise
• Sample effect• SNP/genotype effect• GC content effect
DosageMiner
Black dots: normalized intensity of SNP
Red dots: moving average intensity
Blue line: Deletion, as defined by DosageMiner
DosageMiner
Black dots: normalized intensity of SNP
Red dots: moving average intensity
Green line: Duplication, as defined by DosageMiner
Quality ControlSize matters
Quality ControlSize matters
Quality ControlSample matters
De novo CNVs in trios
• For the 2189 trios, we query the offspring for all deletions and duplications (as reported by DosageMiner) in autosomal chromosomes spanning at least 10 consecutive SNP markers.
• We use the overall variance in SNP intensities for each offspring, to estimate the quality of the intensity data
De novo CNVs in trios
2057 trio-offspring with mean variance <300(arbitrary cutoff value approx. 2SD>mean of all samples)
Chip Trios CNVs 317k 891 17.046DUO 809 15.683370k 357 14.455
De novo CNVs in trios
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 5 10 15 20 25 30 35 40
CNVs/PN
cum
ula
tive
rat
io o
f P
Ns
317k PNs
DUO PNs
370k PNs
De novo CNVs in triosNoise vs. CNVs pr. PN
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 5 10 15 20
CNVs/PN
rati
o o
f tr
ue
CN
Vs
Chr2
De novo CNVs in trios
Chip 317k DUO 370k
PN ratio 0.74 0.77 0.64
CNVs 2028 1589 698
CNV ratio 0.12 0.10 0.05
Using only samples with <10 reported CNVs we retain most of the power (73% of samples) while we get rid of most of the noise.
De novo CNVs in triosPower of detection (n=1500)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 0.0005 0.001 0.0015 0.002 0.0025
population frequency
det
ecti
on
pro
bab
ilit
y
De novo CNVs in trios
Offspring
Father
Mother
Offspring
Father
Mother
De novo deletion or false calling
CNV length Accuracy N CNVs %
10-14 0.42 326 137 0.55
15-19 0.61 69 42 0.17>19 0.89 80 71 0.28
Data from 317k trios, chr1-4
De novo CNVs in trios
Chip 317k DUO 370k
CNVs >9 SNP 2028 1589 698
CNVs >19 SNP 439 333 122
>19 SNP / >9 SNP 0.22 0.21 0.18
De novo CNVs in trios
De novo CNVs in parent-offspring pairs
• We used LOH analysis combined with DosageMiner data to identify de novo deletions in offspring where only one parent is genotyped.
• When 2 or more inheritence errors with genotyped parent were found in a stretch of homozygous markers in offspring, and DosageMiner revealed deletion in offspring and not in genotyped parent we conclude that the deletion is de novo.
De novo CNVs in parent-offspring pairs
Genotyped parent
Offspring
De novo CNVs - results
• We found 70 de novo CNVs at 66 loci• 55 deletions and 15 duplications• 4 deletions observed de novo twice• 20 deletions and 6 duplications observed in
the 1600 vs. 30000 sample• 8 deletions and 2 duplications observed in the
schizophrenia sample• 3 deletions showed nominal association to
schizophrenia
De novo CNVs - resultsChromosome Deletions Duplications CNVs
Chr1 3 1 4 (3)
Chr2 4 2 6
Chr3 4 1 5
Chr4 1 1
Chr5 2 2
Chr6 2 2
Chr7 6 3 9
Chr8 1 1
Chr9 1 1
Chr10 3 3
Chr11 1 1 2
De novo CNVs - resultsChromosome Deletions Duplications CNVs
Chr12 1 1 2
Chr13
Chr14
Chr15 3 2 5 (4)
Chr16 10 (7) 10 (7)
Chr17 4 1 5
Chr18 1 1
Chr19 2 1 3
Chr20 4 (3) 4 (3)
Chr21 1 1
Chr22 3 3
Premutations could provide another mechanism for recurrent CNVs in severe psyciatric disorders.
Father
Mother
Offspring
Complete GW-CNV studies
• Next step is to finish inspecting the 4000 or so candidate CNVs from the 1500 trios to give us a fixed set of confirmed CNVs of >50-100 Kb in >0.001 population frequency.
• This is already well underway, and when finished can be used as a basis for studies in other diseases/traits as well.
Take home message
• Recurrent CNVs may be involved in schizophrenia as well as autism and mental retardation and other severe psychiatric disorders where reduced fecundity is observed
• Analysing CNVdata from GW SNP chips requires much effort to read through the noise.
Thanks to....
• all my coworkers at decode
• the collaborators in SGENE
• EC for funding SGENE
• the participants of the study
• the organisers for inviting me
• you for listening