Lessons from DUF1220 Protein Domains, Cognitive
Disease and Human Brain Evolution II
James M. Sikela, Ph.D.
Department of Biochemistry & Molecular Genetics
Human Medical Genetics and Neuroscience Programs
University of Colorado School of Medicine
Advanced Genome Analysis Course
University of Colorado School of
Medicine
April 23, 2015
Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss
Fortna, et al, PLoS Biol. 2004
Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss
Genes showing
human lineagespecific increases
in copy number
Fortna, et al, PLoS Biol. 2004
DUF1220
Repeat Unit
Popesco, et al, Science 2006
Genome
Human
Chimp
Gorilla
Orangutan
Gibbon
Macaque
Marmoset
Mouse Lemur
Bushbaby
Tarsier
Rabbit
Pika
Mouse
Rat
Guinea Pig
Squirrel
Tree Shrew
Cow
Dolphin
Pig
Horse
Dog
Panda
Cat
Megabat
Microbat
Hedgehog
Shrew
PDE4DIP
2
3
3
4
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Total
DUF1220
272
125
99
92
53
35
31
2
3
1
8
1
1
1
1
1
4
7
4
3
8
3
2
3
1
1
1
1
NBPF
Genes
23
19
15
11
10
10
11
1
2
0
3
0
0
0
1
1
3
3
1
1
3
1
1
2
0
0
0
0
DUF1220 Copy Number in
Mammalian Genomes
- Copy amplification
restricted to anthropoid
species: monkeys, apes
and humans
O’Bleness et al.. G3 Sept (2012).
Sequences Encoding DUF1220 Domains
• Show the largest human lineage-specific increase in
copy number of any protein coding region in the
genome (160 HLS; >270 total in haploid genome)
• Show signs of positive selection especially in primates
• In brain, are expressed only in neurons
• Are highly amplified in human, reduced in great apes,
further reduced in monkeys, single-or-low copy in
prosimians and non-primate mammals, and absent in
non-mammals
• Have increased in human primarily by domain hyperamplification involving DUF1220 triplet
Key Human-Specific Evolutionary Features of 1q21.1 Region
‡*
O’Bleness, et al, Nat Rev
Genet, 2012
1q21.1 Deletions linked to Microcephaly*
1q21.1 Duplications linked to Macrocephaly*
• Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated
with Microcephaly or Macrocephaly and Developmental and
Behavioral Disorders
Brunetti-Pierri, et al, Nature Genetics 2008
• Recurrent Rearrangements of Chromosome 1q21.1 and Variable
Pediatric Phenotypes
Mefford, et al, N. Engl. J. Med. 2008
• *Implies the copy number (dosage) of one or more genes in this
region is influencing brain size in a dose-dependent manner
• These CNVs encompass or are immediately flanked by DUF1220
sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol.,
2009)
DUF1220/NBPF Sequences & Recurrent Disease-associated 1q21.1 CNVs
Dumas, et al, Amer. J. Hum. Gen., 2012
Dumas, et al, Amer. J. Hum. Gen., 2012
Correlation of DUF1220 Copy Number with
Multiple Primate Brain MRI Phenotypes
Is DUF1220 copy number associated with
brain size in humans?
• Custom, high-density 1q21 DNA microarrays to
test copy number variation via arrayCGH
• 42 individuals with 1q21.1 CNVs
• Microcephaly (Type I and Type II Deletions)
• Macrocephaly (Duplications)
• Test for correlation of copy number with head
circumference (FOC Z-score)
• 6 DUF1220 clades & 53 1q21 genes tested
• qPCR validation
1q21 ArrayCGH Profiles for Disease Groups
• Microcephaly
–Class I Deletions
–Class II Deletions
• Larger interval
• More severe
• Macrocephaly
–Duplications
Dumas, et al, Amer. J. Hum. Gen., 2012
Dumas, et al, Amer. J.
Hum. Gen., 2012
BCL9
GRP89A
DUF1220
Copy Number of Genes in the 1q21.1 Region
vs. Brain Size in 5 Primate Species
Human
Chimp
Orangutan
Macaque
Marmoset
Human
Chimp
Orangutan
Macaque
Marmoset
Brain (g)
1350
380
390
88
7
Itga10
1
1
1
1
1
DUF1220*
268
125
92
35
30
Nudt17
1
1
1
1
1
PPIAL4
5
1
1
0
0
Rnf115
1
1
1
1
1
Sec22b
1
1
1
1
1
Cd160
1
1
1
1
1
Notch2nl
1
1
1
1
1
PDZK1
3
1
1
1
1
HFE2
1
1
1
1
1
GPR89
3
1
1
1
1
Txnip
1
1
1
1
1
Hydin
1
0
0
0
0
Polr3gl
1
1
1
1
1
Prkab2
1
1
1
1
1
Polr3
1
1
1
1
1
Pdia3p
1
1
1
1
1
Ankrd34
1
1
1
1
1
Fmo5
1
1
1
1
1
Ankrd35
1
1
1
1
1
Chd1l
1
1
1
1
1
Lix1l
1
1
1
1
1
Bcl9
1
1
1
1
1
Rbm8a
1
1
1
1
1
Acp6
1
1
1
1
1
Gnrhr2
1
1
1
1
1
GJA5
1
1
1
1
1
Pex11b
1
1
1
1
1
GJA8
1
1
1
1
1
*DUF1220 protein domains: NBPF gene family
Dumas, et al, Amer. J. Hum. Gen., 2012
Association of ArrayCGH-predicted DUF1220 Copy Number
with Brain Size (FOC Z score) in 1q21 Disease Population
Dumas, et al, Amer. J. Hum. Gen., 2012
Association of ArrayCGH-predicted DUF1220 Copy Number
& Brain Size (FOC Z score) in Combined Deletion Groups
Dumas, et al, Amer. J. Hum. Gen., 2012
Investigation of DUF1220 Copy Number
in Non-disease Population
• >300 non-disease individuals previously analyzed by
brain sMRI
• 59 DNA samples selected from extremes in gray
matter volume (large [n=29] and small [n=30])
• Analyzed by arrayCGH using custom 1q21 arrays
ArrayCGH Data for Non-Disease Group
DUF1220 CON1 & CON2 Clades
Dumas, et al, Amer. J. Hum. Gen., 2012
Factors that must be reconciled for model linking
1q21.1 instability, evolutionary adaptation &
recurrent disease
• Evolutionary rapid DUF1220 copy number increase
• Estimate, on average, 28 DUF1220 copies added to
human genome every 1 million years since the
Homo/Pan split
• Underlying mechanism must account for
continued, recurrent DUF1220 increases
• Underlying mechanism must account for excess of
1q21.1 disease-associated CNVs
Proposed Mechanism Linking DUF1220,
Brain Evolution, and Disease
Evolutionary
Advantage of
Increase in
Brain Size
1q21 Deletions:
Microcephaly
Schizophrenia
Increase/Retained
1q21.1 Instability
*
1q21.1 Duplications:
Macrocephaly
Autism
Increase in
DUF1220
Copy Number
*Autism, Congenital Heart Disease, Congenital Anom. of Kidney/Urinary Tract, Epilepsy, Intellectual Disability,
Intermittent Explosive Disorder, Macrocephaly, Mayer-Rokitansky-Kuster-Hauser Syn., Microcephaly,
Neuroblastoma, Schizophrenia, Thrombocytopenia-absent-radius (TAR) Syn.
DUF1220 Model
DUF1220 model proposes that:
1) Increasing DUF1220 copy number (dosage)
increases human brain size, and
2) the evolutionary advantage of rapidly increasing
DUF1220 copy number in the human lineage has
resulted in retention of the high genomic instability
of the 1q21.1 region which, in turn, has
precipitated a spectrum of recurrent human brain
and developmental disorders
Dumas & Sikela, Cold Spring Harbor Symposium
Quant. Biol., 2009
Autism & schizophrenia as genomic sister disorders
exhibiting opposite phenotypes
Relative frequencies of copy-number deletions and duplications associated with
autism or schizophrenia
CNV locus
Condition
Deletion Duplication
cases
cases
P-value
(Fisher’s exact)
1q21.1
Autism
Schizophrenia
2
15
10
4
0.001
16p11.2
Autism
Schizophrenia
14
5
5
24
0.00013
22q11.21
Autism
Schizophrenia
1
16
8
1
0.000049
22q13.3
Autism
Schizophrenia
5
0
0
4
0.0079
Boldface italic font indicates that the CNV is documented as a risk factor for the
condition specified.
Adapted from Crespi, Stead &
Elliot, PNAS, 2009
Gaussian distribution of DUF1220 copy number in
human populations: Rich source of overlooked
functional allelic variation
55-90 copies
20-55 copies
140-250 copies
DUF1220 and Autism
• High rates of brain growth in years 0-2
• Greater brain growth -> greater severity
• Strong genetic component to etiology; no major-effect
genes identified.
• 1q21 CNV linked to autism encompasses DUF1220
copies
• DUF1220 copy number not directly examined in
autism/ASD
• Does DUF1220 copy number affect phenotype?
Experimental Design
• 183 Autism Cases
• 135 Controls
• ddPCR used to measure CON1 copy number
• CON1 selected as starting point due to association with
brain size
• Examined differences in overall copy number
between cases and controls
• Examined associations between DUF1220 and
phenotype severity within cases
• Severity determined from Autism Diagnostic InterviewRevised (ADI-R) scores
• Three main measures of severity:
• Impaired social reciprocity
• Impaired communication
• Increased repetitive behaviors
CON1 copy number: ASD vs Controls
Histogram of co$CON1
30
20
Frequency
40
30
0
0
10
10
20
Frequency
50
40
60
70
50
Histogram of asd$CON1
45
50
55
60
65
asd$CON1
CON1
ASD
70
75
80
50
55
60
65
70
75
80
co$CON1
CON1 Controls
• ASD copy number similar to controls
• What about severity?
– DUF1220 has broad copy number range: has the potential to
confer a broad range of phenotypic effect
– CON1 also linked to brain growth (brain growth  severity)
85
Results of Initial Study: CON1 Copy Number
vs ASD Severity
• DUF1220 CON1 dosage is linearly associated with
increasing severity of the primary symptoms of
autism
– Controlled for multiple potential confounders
Davis, et al, PLoS Genetics, 2014
Results of Initial Study: CON1 Copy Number
vs ASD Severity
• DUF1220 CON1 dosage is linearly associated with
increasing severity of the primary symptoms of
autism
– Controlled for multiple potential confounders
– Per single copy increase of CON1:
• 0.25 increase in Social Diagnostic Score (p=0.021)
• 0.18 increase in Communicative Diagnostic Score
(p=0.030)
• 0.10 increase in Repetitive Behaviors Diagnostic
Score (p=0.047)
Davis, et al, PLoS Genetics, 2014
Results of Replication Study: CON1 Copy
Number vs ASD Severity
• Repeated analyses in 166 additional cases
• Per single copy increase in CON1:
– 0.24 increase in Social Diagnostic Score (p=0.036)
– 0.16 increase in Communicative Diagnostic Score
(p=0.07)
– No association with Repetitive Behaviors Diagnostic
Score
Davis, et al, Human Genetics 2015
Autism and DUF1220: Conclusions
• DUF1220 CON1 dosage is linearly related to severity of
symptoms in autism
– more CON1 -> more severe social impairment
– more CON1 -> more severe verbal communication impairment
• Could DUF1220 be involved is autism disease risk?
• Gene sequences important to autism may have been (and
continue to be) missed by conventional genome-wide analyses
– Highly duplicated, copy number polymorphic coding sequences
• Genes important to autism (and schizophrenia) may also be
involved in human brain evolution
– Double-edged sword model (Crow papers and Burns book)
What about DUF1220 and
cognitive aptitude?
• Experimental Design:
• ArrayCGH and ddPCR analysis
• European population; brain MRI measurements
and WISC IQ scores
• New Zealand population; multiple cognitive
measurements including mathematical aptitude
• Looked for associations between CON2 copy
number and cognitive function in each population
DUF1220 copy number is linearly associated with increased
cognitive function as measured by total IQ and mathematical
aptitude scores
-
Davis et al Human Genetics, 2015
(From the abstract):
We identified a linear association between CON2 copy number and cognitive
function in two independent populations of European descent. In North American
males, an increase in CON2 copy number corresponded with an increase in WISC
IQ (R2 = 0.13, p = 0.02), which may be driven by males aged 6–11 (R2 = 0.42, p =
0.003). We utilized ddPCR in a subset as a confirmatory measurement.
This group had 26–33 copies of CON2 with a mean of 29, and each copy increase
of CON2 was associated with a 3.3-point increase in WISC IQ (R2 = 0.22, p =
0.045).
In individuals from New Zealand, an increase in CON2 copy number was associated
with an increase in math aptitude ability (R2 = 0.10 p = 0.018). These were not
confounded by brain size. To our knowledge, this is the first study to report a
replicated association between copy number of a gene coding sequence and
cognitive aptitude. Remarkably, dosage variations involving DUF1220 sequences
have now been linked to human brain expansion, autism severity and cognitive
aptitude, suggesting that such processes may be genetically and mechanistically
inter-related.
Davis et al, Hum Genet 2015
Linear Association of DUF1220 CON2 Copy
Number and Cognitive Aptitude
Fig. 2 Linear association of CON2
with IQ in North American
males. The triangles display
array-based CON2 copy ratio
versus WISC IQ in younger males
(≤10.9). The gray dots display
the same but in older males. The
line is a least squares line of best
fit in the younger male group (R2
= 0.42, p = 0.003, n = 19).
Davis et al, Hum Genet 2015
Summary
DUF1220 domain copy number:
•
•
•
•
•
•
•
•
Largest human-specific increase in copy number of any
protein coding region (~270 human copies)
Human increase accompanied by large disease burden
Highly correlated with brain size (p<1.8x10-6) & cortical
neuron number (p<0.0011) across primate lineages
Highly correlated with brain size in 1q21 disease
population (microcephaly/macrocephaly): (p<1.56x10-7)
Dosage linked with increasing gray matter volume in nondisease population: (CON1:p<0.025); (CON2: p<0.033)
CON2 dosage linked to cognitive aptitude (IQ and math)
Increasing CON1 dosage linked with increasing severity of
social impairment in autism
Suggests DUF1220 dosage may be a key driver of human
brain evolution & cognition, & modulator of autism
severity
A Walk Through Our Genome
--All regions of the genome are not created equal