DUF1220 Domains & the Search for the Genes that Made Us Human James M. Sikela, Ph.D. Human Medical Genetics, Neuroscience, & Comparative Genomics Programs,

DUF1220 Domains & the Search for the Genes that Made Us Human

DUF1220 Domains & the Search for the Genes that Made Us Human

James M. Sikela, Ph.D.Human Medical Genetics, Neuroscience, &

Comparative Genomics Programs,Department of Biochemistry & Molecular Genetics, University of Colorado School of

Medicine

Genomics CourseFebruary 28, 2012

James M. Sikela, Ph.D.Human Medical Genetics, Neuroscience, &

Comparative Genomics Programs,Department of Biochemistry & Molecular Genetics, University of Colorado School of

Medicine

Genomics CourseFebruary 28, 2012

Key PointsKey Points• First gene-based and first genome-wide study of

lineage-specific gene duplication and loss in human and primate evolution

• Dramatic human-specific increase in copy number of DUF1220 protein domains

• DUF1220 copy number linked to evolution of brain size

• Selection of evolutionarily adaptive genome sequences may be driving disease, e.g. 1q21.1

• First gene-based and first genome-wide study of lineage-specific gene duplication and loss in human and primate evolution

• Dramatic human-specific increase in copy number of DUF1220 protein domains

• DUF1220 copy number linked to evolution of brain size

• Selection of evolutionarily adaptive genome sequences may be driving disease, e.g. 1q21.1

Primate EvolutionPrimate Evolution

New World Monkeys (e.g. squirrel monkey,spider monkey)New World Monkeys (e.g. squirrel monkey,spider monkey)

Old World Monkeys (e.g. baboon, rhesus, etc.)Old World Monkeys (e.g. baboon, rhesus, etc.)

GibbonsGibbons

OrangutanOrangutan

GorillaGorilla

HumanHuman

ChimpChimp

BonoboBonoboB/C = ~ 2C/H = ~ 5HC/G = ~ 8HCG/O = ~ 13HCG/O/Gib = ~20Hom/OWM = ~ 25HomOWM/NW = ~ 40

40 MYA

25 MYA

20 MYA

13 MYA

8 MYA

5 MYA

2 MYA

Chimpanzee

Gorilla

Bonobo

Orangutan

More Primates!

---- something has changed!---- something has changed!

Human CharacteristicsHuman Characteristics• Body shape and

thorax• Cranial properties

(brain case and face)• Small canine teeth• Skull balanced upright

on vertebral column• Reduced hair cover• Enhanced sweating• Dimensions of the

pelvis• Elongated thumb and

shortened fingers• Relative limb length

• Body shape and thorax

• Cranial properties (brain case and face)

• Small canine teeth• Skull balanced upright

on vertebral column• Reduced hair cover• Enhanced sweating• Dimensions of the

pelvis• Elongated thumb and

shortened fingers• Relative limb length

• Neocortex expansion• Enhanced language &

cognition• Advanced tool making

• Neocortex expansion• Enhanced language &

cognition• Advanced tool making

modified from S. Carroll, Nature, 2005

Reports of “human-specific” genesReports of “human-specific” genes

• FOXP2– Mutated in family with language disability

• ASPM/MCPH– Mutated in individuals with microcephaly

• HAR1F– Gene sequence highly changed in humans

• DUF1220 protein domains– Highly increased in copy number in

humans; expressed in important brain regions

• FOXP2– Mutated in family with language disability

• ASPM/MCPH– Mutated in individuals with microcephaly

• HAR1F– Gene sequence highly changed in humans

• DUF1220 protein domains– Highly increased in copy number in

humans; expressed in important brain regions

Molecular Mechanisms Underlying Genome Evolution

Molecular Mechanisms Underlying Genome Evolution

• Single nucleotide substitutions- change gene expression &

structure• Genome rearrangements• Gene duplication

- copy number change: gene dosage

- redundancy as a facilitator of innovation

• Single nucleotide substitutions- change gene expression &

structure• Genome rearrangements• Gene duplication

- copy number change: gene dosage

- redundancy as a facilitator of innovation

Gene Duplication & Evolutionary Change

•“There is now ample evidence that gene duplication is the most important mechanism for generating new genes and new biochemical processes that

have facilitated the evolution of complex organisms from primitive

ones.”- W. H. Li in Molecular

Evolution, 1997

•“Exceptional duplicated regions underlie exceptional biology”

- Evan Eichler, Genome Research 11:653-656,

2001

Fig 1. Measuring genomic DNA copy number alteration using cDNA microarrays (array CGH). Fluorescence ratios are depicted in a pseudocolor scale, such that red indicates increased, and green decreased, gene copy number in the test (right) compared to reference sample (left).

Interhominoid cDNA Array-Based Comparative Genomic Hybridization

(aCGH)

Interhominoid cDNA Array-Based Comparative Genomic Hybridization

(aCGH)

Experimental Design Experimental Design

• Carry out pairwise cDNA aCGH comparisons between human and other hominoid species

• Use a >39,000 cDNA microarray representing >29,000 human genes

• Hybridize human genomic DNA (reference sequence: cy3/green) and other hominoid genomic DNAs (test sequence: cy5/red) simultaneously to the microarray

• Visualize aCGH signals “gene-by-gene” along each chromosome across five species: human (n=5), bonobo (n=3), chimpanzee (n=4), gorilla (n=3) and orangutan (n=3)

• Carry out pairwise cDNA aCGH comparisons between human and other hominoid species

• Use a >39,000 cDNA microarray representing >29,000 human genes

• Hybridize human genomic DNA (reference sequence: cy3/green) and other hominoid genomic DNAs (test sequence: cy5/red) simultaneously to the microarray

• Visualize aCGH signals “gene-by-gene” along each chromosome across five species: human (n=5), bonobo (n=3), chimpanzee (n=4), gorilla (n=3) and orangutan (n=3)

Whole Genome Caryoscope Image of Interhominoid aCGH DataWhole Genome Caryoscope Image of Interhominoid aCGH Data

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

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

Summary of Human/Primate ArrayCGH Results

Summary of Human/Primate ArrayCGH Results

• First genome-wide and first gene-based aCGH comparison of human and nonhuman primate gene copy number variation (Fortna, et al 2004)

• 1,004 (4,159) genes identified that showed lineage-specific changes in copy number

• Time machine of evolutionary copy number change

• Gene candidates to underlie lineage-specific traits• Genes identified represent most of major lineage-

specific gene duplications and losses over the last 60 million years of human and primate evolution (Dumas, et al 2007)

• First genome-wide and first gene-based aCGH comparison of human and nonhuman primate gene copy number variation (Fortna, et al 2004)

• 1,004 (4,159) genes identified that showed lineage-specific changes in copy number

• Time machine of evolutionary copy number change

• Gene candidates to underlie lineage-specific traits• Genes identified represent most of major lineage-

specific gene duplications and losses over the last 60 million years of human and primate evolution (Dumas, et al 2007)

0 50 100 140 170 2501p36 1p34 1p31 1p22 1q21 1q23 1q32 1q41Mb

1p1320 210

1

2

0 90 130 170 200 2402p24 2p11 2q14 2q31 2q33 2q37Mb 2q2130 110 2p1650

3

Mb 0 80 160 180 2003p25 3p12 3q13 3q263q2520 130 3p21 50 3q21 3q28

Mb 0 100 1904p16 4q24 4q12 4q3410 80 4p12 50 4q31

4

140

Mb

5

0 100 1905p15 5q23 5q1320 70 5q1150 5q34150130

Mb

6

0 50 1706p25 6q126p21 30 130 10 6p22 40 6q1490 6q22 6q25

Mb

7

0 100 1607p21 7q11 7q21 60 30 7p14 90 7q31130 7q35140 7q22

Mb

8

0 120 1508p21 8q12 40 20 8p12 80 8q21100 8q2460

Mb

9

120 1509p23 9q21 40 30 9p13 80 9q22 100 9q34600

120 14010p15 10q21 10q24 40 2010p11 80 100 10q26500Mb

10

10q25

Mb

11

90 14011p15 11q12 11q13 20 10 11p14 70 11q24500 80 120 11q22 11q14

1 2 3

12

Mb 110 13012p13 12q13 12q14 30 10 12p12 70 12q24500 90 12q21

13

Mb 13q12 13q21 13q33 3013q14 110500 90

14

Mb 14q11 14q31 50 3014q13 700 1009014q32

15

Mb 15q13 700 10015q26 20 40 5015q21 15q2415q22

4 5

5q15

8q22

9q33

14q22

6 7

8

9

10

11

12 13

14

15

16Mb

16p13 700 9016q24 10 20 3016p12 16q22 5016q12

17p13 70 9017q23 10 20 3017q11 17q21 5017q12 17q25

17Mb

18Mb

18p110 10 20 8018q12 5018q21

19Mb

19p13 500 60 10 20 40 19q12 19q13 19p11

20Mb

20p130 10 30 6020q11 5020q13 20

21Mb

0 30 40 5021q22

22Mb

0 3022q11 50 22q13 20 40

XMb

Xp220 50 150Xp11 130Xq21 20 100 70 Xq26 Xq28

YMb

0Yp11 50 20

19q11

16

17

18

19

20

21 22

23

2>_0.5<_

Test/Reference ratio:

1

Human (Homo Sapiens)Bonobo (Pan Paniscus)Chimpanzee (Pan Troglodytes)

Orangutan (Pongo Pygmaeus)Gorilla (Gorilla Gorilla)

3

H

B

C

G

O

6

H

B

C

G

O

9

H

B

C

G

O

13

H

B

C

G

O

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

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

““This (Fortna, et al, 2004) is the first This (Fortna, et al, 2004) is the first time that copy number changes among time that copy number changes among

apes have been assayed for the vast apes have been assayed for the vast majority of human genes, and we can majority of human genes, and we can

expect that the biological consequences expect that the biological consequences of the 140 human-specific copy number of the 140 human-specific copy number changes identified in this study will be changes identified in this study will be heavily investigated over the coming heavily investigated over the coming

years. “years. “

---M. Hurles, ---M. Hurles, PLoS BiolPLoS Biol. 2004. 2004

DUF1220Repeat Unit

Popesco, et al, Science 2006

InterPro-predicted DUF1220-containing proteins (NBPF family*)

*Vandepoule, et al, Mol. Biol. & Evol, 2005*Vandepoule, et al, Mol. Biol. & Evol, 2005

Copy Number of DUF1220 (Q8IX62/17-33) Sequences in Primate Species

0

10

20

30

40

50

60

70

Q-P

CR

Pre

dic

ted

Co

py

Nu

mb

er

70

60

50

40

30

20

100

Q-P

CR

Pre

dic

ted

Co

py

N

um

ber

Copy Number of DUF1220 (Q8IX62/17-33)Sequences in Primate Species

Hu

ma

n

Bo

no

bo

Ch

imp

Go

rill

a

Ora

ng

uta

n

Gib

bo

n

Mac

aqu

e

Bab

oo

n

Summary of aCGH, Q-PCR and BLAT results:

Summary of aCGH, Q-PCR and BLAT results:

• DUF1220 domains are highly amplified in human, reduced in great apes, further reduced in Old & New World monkeys, single or low copy non-primate mammals and absent in non-mammals

• DUF1220 domains are highly amplified in human, reduced in great apes, further reduced in Old & New World monkeys, single or low copy non-primate mammals and absent in non-mammals

DUF1220 copy number in Animal Genomes

Genome PDE4DIP

DUF1220

Total

DUF1220

NBPF

genes

Human 2 268 21

Chimp 3 125 15

Gorilla 3 99 15

Orangutan 4 92 11

Macaque 1 35 10

Marmoset 1 30 10

Rabbit 1 8 3

Mouse 1 1 0

Rat 1 1 0

Guinea Pig 1 1 0

Genome PDE4DIP

DUF1220

Total

DUF1220

NBPF

genes

Cow 1 6 2

Pig 1 3 1

Horse 1 8 3

Dog 1 3 1

Panda 1 2 1

Opposum 1 1 0

EuarchotanglinesEuarchotanglines LaurasiatheriaLaurasiatheria

AfrotheriaAfrotheria

MetatheriaMetatheria

Elephant 1 1 1

PrototheriaPrototheriaPlatypus 1 1 0

Chicken 0 0 0

Lizard 0 0 0

Frog 0 0 0

Zebrafish 0 0 0

Other VertebratesOther VertebratesA total of 40 genomes were A total of 40 genomes were searched, but only the 22 with 4X searched, but only the 22 with 4X coverage or higher are displayed.coverage or higher are displayed.

DUF1220 Copy Number Statistics in hg19 build

DUF1220 Copies

Total in Human Genome 272

Total amplified HLS DUF1220 Triplets 129

Total DUF1220 in Last Common Ancestor of Homo/Pan 102

Total of Newly Added Copies in Human Lineage 167

Total Copies Added via Domain Amplification 146

Total Copies Added via Gene Duplication 21

Average Number Added to Human Lineage every million years 28

This table shows the unprecedented DUF1220 copy number increase in the human lineage. The primary mechanism for this expansion was domain amplification via hyper-amplification of the HLS DUF1220 triplet.

Sequences encoding DUF1220 domains

Sequences encoding DUF1220 domains

• Show a major copy number burst in primates

• Are increasingly amplified generally as a function of a species evolutionary proximity to humans, where the greatest number of copies (270) is found

• Show signs of positive selection• Are highly expressed in brain regions

associated with higher cognitive function• In brain show neuron-specific expression

preferentially in cell bodies and dendrites

• Show a major copy number burst in primates

• Are increasingly amplified generally as a function of a species evolutionary proximity to humans, where the greatest number of copies (270) is found

• Show signs of positive selection• Are highly expressed in brain regions

associated with higher cognitive function• In brain show neuron-specific expression

preferentially in cell bodies and dendritesPopesco, et al, Science 2006Popesco, et al, Science 2006

•Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Abnormalities

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

•Recurrent Reciprocal 1q21.1 Deletions and Duplications Associated with Microcephaly or Macrocephaly and Developmental and Behavioral Abnormalities

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

1q21.1 Deletions* Linked to Microcephaly

1q21.1 Duplications* Linked to Macrocephaly

1q21.1 Deletions* Linked to Microcephaly

1q21.1 Duplications* Linked to Macrocephaly

We note that these CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009)

We note that these CNVs encompass or are immediately flanked by DUF1220 sequences (Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009)

*Implies human brain size directly related to the dosage of one or more genes in these 1q21.1 CNVs*Implies human brain size directly related to the dosage of one or more genes in these 1q21.1 CNVs

DUF1220/NBPF Sequences & Recurrent Disease-associated 1q21.1 CNVs

Association (p<0.0001) of human head circumference (FOC Z-score) & DUF1220 copy

number

Association (p<0.0001) of human head circumference (FOC Z-score) & DUF1220 copy

number

Head Circumference (FOC Z-Score) vs. DUF1220 Copy Number

-6

-4

-2

0

2

4

6

20 30 40 50 60 70 80

Q-PCR-Predicted DUF1220 Copy Number

FO

C Z

-Sc

ore

Class II Deletion

Class I Deletion

Duplication

Copy number of genes in the 1q21.1-q21.2 region versus brain size

•46 1q21.1 genes compared along with brain size across 5 primate species

•DUF1220 shows the most

dramatic human-specific copy number increase.

•The evolutionary increase in DUF1220 copy number parallels the increase in brain size.

  Human Chimp Orangutan Macaque MamosetBrain Size (g) 1350 380 390 88 7Copy #DUF1220 272 125 92 35 30PPIAL4 5 1 1 0 0LOC728855 5 2 2 2 1FAM72D 2 0 0 0 0SRGAP 1 0 0 0 0PDE4DIP 3 3 4 1 1SEC22B 1 1 1 1 1NOTCH2NL 1 1 1 1 1HFE2 1 1 1 1 1TXNIP 1 1 1 1 1POLR3 2 2 2 2 2ANKRD34 1 1 1 1 1ANKRD35 1 1 1 1 1LIX1L 1 1 1 1 1RBM8A 1 1 1 1 1GNRHR2 1 1 1 1 1PEX11B 1 1 1 1 1ITGA10 1 1 1 1 1NUDT17 1 1 1 1 1RNF115 1 1 1 1 1CD160 1 1 1 1 1PDZK1 3 1 1 1 1GPR89 3 1 1 1 1PRKAB2 1 1 1 1 1PDIA3P 1 1 1 1 1FMO5 1 1 1 1 1CHD1L 1 1 1 1 1BCL9 1 1 1 1 1ACP6 1 1 1 1 1GJA5 1 1 1 1 1GJA8 1 1 1 1 1LOC645166 1 0 0 0 0FCGR1 2 1 1 1 1SV2A 1 1 1 1 1BOLA1 1 1 1 1 1MTMR11 1 1 1 1 1OTUD7B 1 1 1 1 0SF3B4 1 1 1 1 1VPS45 1 1 1 1 1PLEKHO1 1 1 1 1 1ANP32E 1 1 1 1 1PRPF3 1 1 1 1 1C1orf54 1 1 1 1 1MRPS21 1 1 1 1 1CA14 1 1 1 1 1C1orf51 1 1 1 1 1APH1A 1 1 1 1 1

DUF1220 Copy Number Versus Brain Size

* Neandertal DUF1220 copy number is estimate based on sequence read depth from the Neandertal genome (Green et al 2010).

-but correlation is not causation

Factors that must be reconciled with model linking 1q21.1 instability, evolutionary

adaptation & recurrent disease

Factors that must be reconciled with model linking 1q21.1 instability, evolutionary

adaptation & recurrent disease

• Evolutionarily rapid DUF1220 copy number increase– Estimate, on average, 28 more DUF1220

domains added to human genome every 1 million years since Homo/Pan split

• Underlying mechanism must account for continued, recurrent DUF1220 increases

• Underlying mechanism must account for excess of 1q21.1 disease-associated CNVs containing dosage-sensitive genes

• Evolutionarily rapid DUF1220 copy number increase– Estimate, on average, 28 more DUF1220

domains added to human genome every 1 million years since Homo/Pan split

• Underlying mechanism must account for continued, recurrent DUF1220 increases

• Underlying mechanism must account for excess of 1q21.1 disease-associated CNVs containing dosage-sensitive genes

Increased 1q21.1 Instability

Increased 1q21.1 Instability

Increase inDUF1220

Copy Number

Increase inDUF1220

Copy Number

Evolutionary Advantage(Increase in Brain Size?)

Evolutionary Advantage(Increase in Brain Size?)

Proposed Mechanism Linking DUF1220, Brain Evolution and

Disease

Proposed Mechanism Linking DUF1220, Brain Evolution and

Disease1q21.1 duplications

Macrocephaly; Autism*

1q21.1 duplications

Macrocephaly; Autism*

1q21.1 deletions

Microcephaly; Schizophrenia*

1q21.1 deletions

Microcephaly; Schizophrenia*

*Diseases proposed as “Diametric Opposites” (including brain size), Crespi, Stead & Elliot, PNAS, 2009

*Diseases proposed as “Diametric Opposites” (including brain size), Crespi, Stead & Elliot, PNAS, 2009

DUF1220 Model*DUF1220 Model*

DUF1220 model proposes that: 1) DUF1220 copy number is directly involved

in influencing human brain size, and2) the evolutionary advantage of rapidly

increasing DUF1220 copy number in the human lineage has resulted in favoring 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) DUF1220 copy number is directly involved

in influencing human brain size, and2) the evolutionary advantage of rapidly

increasing DUF1220 copy number in the human lineage has resulted in favoring 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*Dumas & Sikela, Cold Spring Harbor Symposium Quant. Biol., 2009

Concluding ThoughtsConcluding Thoughts• DUF1220 domains shows the largest HLS DUF1220 domains shows the largest HLS

protein coding copy number increase in the protein coding copy number increase in the genome genome – But no one gene made us humanBut no one gene made us human– DUF1220 genotyping challengesDUF1220 genotyping challenges

• We know more about our genome than everWe know more about our genome than ever– But there are vast areas of our genome But there are vast areas of our genome

about which we know virtually nothingabout which we know virtually nothing– No mammalian genome has been No mammalian genome has been

completely sequencedcompletely sequenced

AcknowledgementsAcknowledgements• Sikela Lab• Laura Dumas • Majesta O’Bleness• Maggie Popesco• Erik MacLaren• Andy Fortna • Jan Hopkins• Jonathon Keeney• Jack Davis• Jay Jackson• Megan Sikela• Michael Cox• Kriste Marshall• Matt Brenton• Sonya Burgers• Raquel Hink• Erin Dorning• Park McNair

• Sikela Lab• Laura Dumas • Majesta O’Bleness• Maggie Popesco• Erik MacLaren• Andy Fortna • Jan Hopkins• Jonathon Keeney• Jack Davis• Jay Jackson• Megan Sikela• Michael Cox• Kriste Marshall• Matt Brenton• Sonya Burgers• Raquel Hink• Erin Dorning• Park McNair

• Collaborators• Stanford

– Jon Pollack– Young Kim

• Univ. of Kansas - Gerald Wyckoff

• Univ of Utah– Lynn Jorde

• Baylor College– Pawel Stankiewicz– Sau Wai Cheng

• UCSOM– Epidemiology

• Tasha Fingerlin– Preventive Medicine &

Biometrics• Anis Karimpour-Fard

– Neuroscience Program• Rock Levinson• John Caldwell

• Collaborators• Stanford

– Jon Pollack– Young Kim

• Univ. of Kansas - Gerald Wyckoff

• Univ of Utah– Lynn Jorde

• Baylor College– Pawel Stankiewicz– Sau Wai Cheng

• UCSOM– Epidemiology

• Tasha Fingerlin– Preventive Medicine &

Biometrics• Anis Karimpour-Fard

– Neuroscience Program• Rock Levinson• John Caldwell

A Walk Through Our GenomeA Walk Through Our Genome

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