Lessons from DUF1220 Protein Domains, Cognitive Disease and Human Brain Evolution II James M. Sikela, Ph.D. Department of Biochemistry & Molecular Genetics

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 ProgramsUniversity of Colorado School of Medicine

Advanced Genome Analysis CourseUniversity of Colorado School of

Medicine April 23, 2015

Fortna, et al, PLoS Biol. 2004Fortna, et al, PLoS Biol. 2004

Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

Fortna, et al, PLoS Biol. 2004Fortna, et al, PLoS Biol. 2004

Human & Great Ape Genes Showing Lineage-Specific Copy Number Gain/Loss

Genes showing human lineage-specific increases in copy number

DUF1220Repeat Unit

Popesco, et al, Science 2006

Genome PDE4DIPTotal

DUF1220NBPFGenes

Human 2 272 23Chimp 3 125 19Gorilla 3 99 15Orangutan 4 92 11Gibbon 3 53 10Macaque 1 35 10Marmoset 1 31 11Mouse Lemur 1 2 1Bushbaby 1 3 2Tarsier 1 1 0Rabbit 1 8 3Pika 1 1 0Mouse 1 1 0Rat 1 1 0Guinea Pig 1 1 1Squirrel 1 1 1Tree Shrew 1 4 3Cow 1 7 3Dolphin 1 4 1Pig 1 3 1Horse 1 8 3Dog 1 3 1Panda 1 2 1Cat 1 3 2Megabat 1 1 0Microbat 1 1 0Hedgehog 1 1 0Shrew 1 1 0

O’Bleness et al.. G3 Sept (2012).

DUF1220 Copy Number in Mammalian Genomes

-Copy amplification restricted to anthropoid species: monkeys, apes and humans

Sequences Encoding DUF1220 Domains• Show the largest human lineage-specific increase in Show the largest human lineage-specific increase in

copy number of any protein coding region in the copy number of any protein coding region in the genome (160 HLS; >270 total in haploid genome) genome (160 HLS; >270 total in haploid genome)

• Show signs of positive selection especially in primatesShow signs of positive selection especially in primates• In brain, are expressed only in neuronsIn brain, are expressed only in neurons• Are highly amplified in human, reduced in great apes, Are highly amplified in human, reduced in great apes,

further reduced in monkeys, single-or-low copy in further reduced in monkeys, single-or-low copy in prosimians and non-primate mammals, and absent in prosimians and non-primate mammals, and absent in non-mammalsnon-mammals

• Have increased in human primarily by domain hyper-Have increased in human primarily by domain hyper-amplification involving DUF1220 tripletamplification 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


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

• Microcephaly–Class I Deletions–Class II Deletions

• Larger interval• More severe

• Macrocephaly–Duplications

1q21 ArrayCGH Profiles for Disease Groups



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


Association of ArrayCGH-predicted DUF1220 Copy Number with Brain Size (FOC Z score) in 1q21 Disease Population


Association of ArrayCGH-predicted DUF1220 Copy Number & Brain Size (FOC Z score) in Combined Deletion Groups


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


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

Increase in DUF1220 Copy

Number

1q21 Deletions:MicrocephalySchizophrenia

1q21.1 Duplications:MacrocephalyAutism

Proposed Mechanism Linking DUF1220, Brain Evolution, and Disease

*

*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.

Evolutionary Advantage ofIncrease inBrain Size

Increase/Retained1q21.1 Instability

DUF1220 ModelDUF1220 Model

DUF1220 model proposes that: 1) Increasing DUF1220 copy number (dosage)

increases human brain size, and2) 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

DUF1220 model proposes that: 1) Increasing DUF1220 copy number (dosage)

increases human brain size, and2) 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., 2009Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009

Relative frequencies of copy-number deletions and duplications associated with autism or schizophrenia

CNV locus Condition Deletion Duplication P-value cases cases (Fisher’s exact)

1q21.1 Autism 2 10 0.001Schizophrenia 15 4

16p11.2 Autism 14 5 0.00013Schizophrenia 5 24



Boldface italic font indicates that the CNV is documented as a risk factor for thecondition specified.

Relative frequencies of copy-number deletions and duplications associated with autism or schizophrenia

CNV locus Condition Deletion Duplication P-value cases cases (Fisher’s exact)


16p11.2 Autism 14 5 0.00013Schizophrenia 5 24



Boldface italic font indicates that the CNV is documented as a risk factor for thecondition specified. Adapted from Crespi, Stead &

Elliot, PNAS, 2009Adapted from Crespi, Stead & Elliot, PNAS, 2009

Autism & schizophrenia as genomic sister disorders exhibiting opposite phenotypes

Autism & schizophrenia as genomic sister disorders exhibiting opposite phenotypes

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 Interview-Revised (ADI-R) scores

• Three main measures of severity:• Impaired social reciprocity• Impaired communication• Increased repetitive behaviors

CON1 copy number: ASD vs Controls

CON1 ASD

• 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)

CON1 Controls

• DUF1220 CON1 dosage is linearly associated with increasing severity of the primary symptoms of autism– Controlled for multiple potential confounders

Results of Initial Study: CON1 Copy Number vs ASD Severity

Davis, et al, PLoS Genetics, 2014

• 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)

Results of Initial Study: CON1 Copy Number vs ASD Severity

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

We identified a linear association between CON2 copy number and cognitive function in two independent populations of European descent. In North Americanmales, 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.

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):

Davis et al, Hum Genet 2015

Linear Association of DUF1220 CON2 Copy Number and Cognitive Aptitude

Davis et al, Hum Genet 2015

Fig. 2 Linear association of CON2 with IQ in North Americanmales. 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).

SummaryDUF1220 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 non-

disease 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 GenomeA Walk Through Our Genome

--All regions of the genome are not created equal--All regions of the genome are not created equal--All regions of the genome are not created equal--All regions of the genome are not created equal

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Lessons from DUF1220 Protein Domains, Cognitive Disease and Human Brain Evolution II James M. Sikela, Ph.D. Department of Biochemistry & Molecular Genetics