2. - NAS-Sites.org

Genomic disorders, mechanisms for
copy number variation,
variation & CNV in evolution
Exploring Human Genomic Plasticity &
Environmental Stressors: Emerging
Evidence
E
id
on Telomeres,
T l
Copy
C
Number Variation & Transposons
National Academies of Science
Washington, D.C
4 October 2012
J
James
R
R. L
Lupski,
ki M
M.D.,
D Ph
Ph.D.,
D D
D.Sc
S
Department of Molecular & Human Genetics
& Department of Pediatrics
Baylor College of Medicine
& Texas Children’s Hospital
Houston, TX
http://www.bcm.edu/geneticlabs/
Topics to be discussed:
1) Background – CNV & gene dosage
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &
other highly complex genomic changes
5) CNV & evolution, environmental mutagenesis
Interpersonal Genome Variation:
( germ-line genomic variation )
1)) Background
g
– CNV & g
gene dosage
g
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &CGR
(somatic intercellular variation)
5) CNV & evolution, environmental mutagenesis
CNV & phenotypes; an historical overview
Phenotypic Variation in Datura
Due to Changes in
Chromosome Copy Number
The American Naturalist
Vol. 56 : 16-31, 1922
Dr. Albert Francis Blakeslee
S i for
Station
f experimental
i
l
evolution
Cold Spring Harbor L.I.,
LI
N.Y.
Calvin B. Bridges (1936)
The Bar “Gene”
Gene a duplication
duplication.
Science 83:210-211
“the ‘puff’…is more pronounced;
the banding is more discontinuous
…synapsis is disturbed”
duplication
triplication
DUP and TRP convey distinct phenotypes
“The respective shares attributable
in tthe
e tota
total e
effect
ect to tthe
e
genic balance change [i.e. dosage]
and to the
position-effect change
seems to be at present a matter
of taste”
taste
- Calvin Bridges
Genic‐Scale Rearrangements & Human Di
Disease: A Historical Perspective
A Hi t i l P
ti
α‐globin duplication
α‐globin duplication
β‐thalassemia
(mild)
Red‐green color blindness
Higgs, et al. (1980) Nature
284:632‐5.
Nathans, et al. Nathans,
et al.
(1986) Science
232:203‐10
Glucocorticoid‐remediable Lifton, et al. (1992) Nature
aldosteronism (hypertension)
ld t
i (h
t i ) (1992) Nature
355:262‐5
Genomic rearrangements? APP duplication and early‐onset Alzheimer disease
d
l
t Al h i
di
Delabar, et al. (1987) Science 235:1390‐2
5
Quantification
Gene Dosage
Genee Dosage
4
3
2
1
Southern to determine gene dosage
d
0
APP
Control Loci
Two reports then argued against APP
d l
duplication in Alzheimer disease
lh
d
Tanzi, et al. (1987) Science 238:666‐9
<10 patients each
<10 patients each
Podlisny, et al. (1987) Science 238:669‐71
7 pages of negative data published; > 2X the 3 pages of positive data
APP duplication and early‐onset Alzheimer disease again!
Alzheimer disease…again!
20 years later !!!
QMPSF
In 5 families with autosomal dominant early onset Alzheimer disease
Rovelet ‐ Lecrux, et al. (2006) Nat Genet 38:24‐26
Molecular mechanisms for chromosomal
syndromes,
y
, Mendelian dz and complex
p
traits.
A. Alzheimer disease
a) trisomy 21
b) copy number variation
duplication of APP
c) SNPs in promoter regions
promoters
SNPs
APP
APP coding exons
APP coding exons
+ genic pt mut !
B. Parkinson disease
b) SNPs in promoter regions
a) copy number variation
triplication of SNCA
promoters
duplication of SNCA
SNPs
SNCA
SNCA coding exons
Shiga, Inoue, & Lupski (2007) Mendelian, non‐Mendelian,
Multigenic inheritance and complex traits. In, The Molecular and Genetic Basis of Neurological and Psychiatric Disease
Rosenberg,, Prusiner, DiMauro, Barchi, (Eds.)
Human genome variation and disease:
heuristic models to investigate genetic architecture of disease >1 gene in CNV
contributes to
phenotype?
C ti
Contiguous
Gene
Syndrome?
digenic &
triallelic
Aneuploidy =
a big CNV!
J.R. Lupski, J.W. Belmont, E. Boerwinkle, R.A. Gibbs (2011) Cell 147: 32‐43
Interpersonal Genome Variation:
1) Background – CNV & gene dosage
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &
other highly complex genomic changes
5) CNV & evolution, environmental mutagenesis
Mechanisms for genomic disorder associated
human genome rearrangements
Feng Zhang
NAHR
NHEJ
MMBIR: microhomologymediated, break induced
p
replication
FoSTeS
FoSTeS ×1
MEI – mobile
element insertion
L1 Retrotransposition
FoSTeS × 2
TS
P
OH
TSD
RECOMBINATION
TSD
REPLICATION
Zhang, Gu, Hurles, Lupski (2009) Ann Rev Genomics and Hum Genet 19:451-481
The CMT1A duplication – a CNV paradigm
Raeymakers,
y
, Timmerman,, et al. ((1991)) Neuromuscular Disorders 1 :93-97
Lupski, et al. (1991) Cell 66 :219-232 [duplication] Lupski, et al (1992) Nat Genet
1:29-33 [gene dosage] ; Pentao, Liu, et al (1992) Nat Genet 2 :292-300 [NAHR]
Proximal
CMT1A-REP
Distal
CMT1A-REP
B
A
C
~ 70% of all CMT1 pts
D
76-90% of sporadic CMT1
[de novo mutation]
PMP22
NORMAL: PMP22 = 2n
B’
A’
C’
D’
CMT1A: PMP22 = 3n
HNPP:
PMP22 = 1n
A
CNV dzs:
Schizophrenia
Autism
Obesity
B
C
B’
C’
JCT
CMT1A DUPLICATION
D’
A’
D
JCT
HNPP DELETION
Mechanism for deletion &
reciprocal
i
l duplication
d li ti
L. Potocki & J. R. Lupski
p
NORMAL
NORMAL:
“I LIKE TO SWIM IN THE OCEAN BUT I DO
NOT LIKE TO SWIM IN THE POOL
POOL..”
DELETION:
“I LIKE
IKE TO SWIM IN THE POOL
POO .””
POOL.”
DUPLICATION:
DUPLICATION
“I LIKE TO SWIM IN THE OCEAN BUT I DO NOT
LIKE TO SWIM IN THE OCEAN BUT I DO NOT
LIKE TO SWIM IN THE POOL
POOL.”
.”
Genomic Disorders
Concept predicated on two major premises:
- genomic
i rearrangements
t NOT
sequence
q
based changes
g
- genome architecture incites
genome instability
Lupski (1998) Genomic Disorders: Structural features of the
human genome can lead to DNA rearrangements and human
disease traits Trends in Genetics 14:415
14:415-420
420
Lupski (2009) Genomic Disorders: ten years on
Genome Medicine 1:42.1-42.11
Calvin B. Bridges (1936)
The Bar “Gene”
Gene a duplication
duplication.
Science 83:210-211
“the ‘puff’…is more pronounced;
the banding is more discontinuous
…synapsis is disturbed”
duplication
triplication
TRP – occurs de novo? OR from pre-rexisting DUP?
mild
CMT1A
BAB3328
8
severe
CMT1A
BAB3
3330
mild
CMT1A
BAB
B3329
BAB33
331
CMT1A duplication becomes a triplication in a family (unpublished)
Shay Ben-Shachar & Avi Orr-Urtreger; Tel Aviv
NAHR as the mechanism for recurrent genomic rearrangements
genomic rearrangements A
C
*
*
*
duplication
deletion
B
interchromosome
d l ti
deletion
duplication
recurrent translocation map
inversion
intrachromosome
(interchromatid)
d l ti
deletion
intrachromatid
d l ti
deletion
sister chromatid
exchange
isochromosome
duplication
Liu, et al. (2012) Curr Opin in Gen and Develop 22:211-220
iso17q –somatic
isoY & isoX - constit.
Genomic disorders:
a new discipline of medical genetics
a new discipline of medical genetics
Post-genomic era
Bridges (1936) Lupski et al. (1991)
Science 83:210‐211
Cell 66:219‐232
The Bar “Gene” duplication Genomic duplication causes causes an eye phenotype
causes an eye phenotype neurological disease
neurological disease
1936
Courtesy Dr Pengfei Liu
1991
1998
genomic
disorders
defined
Cheung et al. (2005)
Genet. Med. 7:422‐432
Clinical utilization of CGH
Feb ’04
Clinical
CMA
2005
N = 45,894;29FEB2012 rare dz day!
N > 50,000 today!!!
Baylor Array CGH Team- clinically introduced high
resolution human genome analyses Feb’04
Clinical Cytogeneticists Clinical Development
Ankita Patel, Ph.D.
Patricia Hixson, Ph.D.
Cheerleaders
Sau wai Cheung, Ph.D.
Sisi Bi, B.S.
Jim Lupski, M.D., Ph.D., D.Sc.
Carlos Bacino, M.D.
Marcus Coyle B.S., M.A.
Art Beaudet, M.D.
Pawel Stankiewicz, M.D., Ph.D.
Rodger Song, B.S.
Seema Lalani, M.D.
Rebecca Davis, B.S.
General Manager
Weimin Bi, Ph.D.
Lu Yang, B.S.
Sean Kim, M.B.A.
Amy Breman, PhD.
Amanda Fullerton, B.S.
Robert Johnson, Ph.D.
Genetic Counselors
Mitochondrial Disease
Arrays
Patricia Ward, M.S.
Sandra Peacock, M.S.
Lee-Jun Wong, Ph.D.
Administration
Jeff Mize, M.B.A., C.P.A.
Marketing
Alicia Braxton, M.S.
Array Development
Mike Frazier, B.S.
Laura Ellis, M.S
Pawel Stankiewicz, M.D., Ph.D.
T. Brandon Perthuis, B.S.
Statistics/Bioinformatics Tomek Gambin, B.S.
Aloma Geer, Ph.D.
Chad Shaw, Ph.D.
Eric Burgess, B.S.
Aleksandar Milosavljevic, Ph.D.
Jian Li, B.S.
Prenatal Genetics
Christine Eng, M.D.
Ignatia Van Den Veyver, M.D.
CMA. - chromosomal
C
microarray analyses
N = 50,310 (19 Aug 2012)
Genome‐wide CNV studies in patients:
lessons learnt Apparently Simple
lessons learnt
A locus can be subject to recurrent
A
locus can be subject to recurrent or
non‐recurrent events.
All rearrangement mechanisms possible at a locus, but particular type may be favored by local genome architecture.
y
g
Gains (dup, trp) losses (del) and complexities can occur.
Diseases are often sporadic due to de novo mutations.
New mutations are quite frequent for CNV (10‐6 6 to 10‐44) compared to SNV (10‐8) [100X – 10,000X !!!].
Potocki-Lupski syndrome (PTLS;MIM #610883)
2000, seven patients with common
duplication were described;
2007 multidisciplinary
2007,
ltidi i li
clinical
li i l study
t d
Definition of a genomic disorder – from
mechanism to clinical delineation
Potocki et al (2000) Nat Genet 24:84-87
Potocki et al (2007) AJHG 80:633-649
PTLS Family Conference July 2012 p
,
Texas Children’s Hospital Houston,TX
smile – say cheese!
silly face!
> 300 patients with PTLS in families’ database
Rearrangement frequency at 17p11.2
Feng Zhang
FAMILIES
STUDIED:
PTLS duplication
PTLS
d li ti
N=79
SMS deletion
N=131
Pengfei Liu
Distribution of different mechanisms in del/dup Recurrent
(NAHR)
Nonrecurrent simple
(FoSTeS or NHEJ)
Complex (FoSTeS or multiple NHEJ)
Total
Deletion
107 (81.7%)
21 (16.0%)
3 (2.3%)
131
Duplication
56 (70.9%)
9 (11.4%)
14 (17.7%)
79
N = 210 pts!
What have we learned?
i) NAHR mediated recurrent rearrangements account for majority of the events.
ii) An additional LCR‐mediated uncommon recurrent event (UR2) was identified
ii) Deletion : duplications :: 2:1, for de novo NAHR. Turner et al. (2008) Nat. Genet.
iii) Complex rearrangements is more prevalent (~ 8X !) in duplications.
Six types of recurrent rearrangements at 17p11.2
all three LCRs are similar in identity; ~98.6% !
What makes one recurrent rearrangement more prevalent than another?
i.e. what determines the NAHR frequency at a locus?
Inter-LCR
distance
LCR
length
NAHR mediated rearrangement frequency at a given locus correlates positively with LCR length & inversely influenced by inter‐LCR distances
inter‐LCR distances
LCR Length
LCR Distance
LCR Length (Kb)
LCR Length
log (Fre
equency)
ln (Freq
quency)
Legend:
Common
Recurrent
Uncommon
Recurrent 1
LCR distance
Uncommon
Recurrent 2
Red: Dup
Green: Del
NAHR
Synapsis
‐‐ Alignment of homologues in meiosis
Terry Hassold Lab
Petrice et al., (2005) Meiotic Synapsis Proceeds from a
Limited Number of Subtelomeric Sites in the Human Male.
Am J Hum Genet. 77: 556-566.
Ectopic synapsis as a mediator of
ectopic crossing over ?
ectopic crossing over ? Pengfei
Liu *
Liu, et al. (2011) Am J Hum Genet 89:580-588
* 2012 Cotterman Award
c/w: i) NAHR & AHR hotspots coincide
ii) same PRDM9 hotspot motif used
iii) yst synaptonemal complex mutants abolish ectopic HR!!! Shinohara Lab
Evidence in yeast
Depletion of Zip1, an essential component of yeast synaptonemal Depletion
of Zip1 an essential component of yeast synaptonemal
complex, almost completely abolishes ectopic crossingover
(Shinohara et al., personal communication)
Topics to be discussed:
1) Background – CNV & gene dosage
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &
other highly complex genomic changes
5) CNV & evolution, environmental mutagenesis
Mechanisms for genomic disorder associatred
human genomic rearrangements
Feng Zhang
NAHR
NHEJ
MMBIR: microhomologymediated, break induced
p
replication
FoSTeS
FoSTeS ×1
MEI – mobile
element insertion
L1 Retrotransposition
FoSTeS × 2
TS
P
OH
TSD
RECOMBINATION
TSD
REPLICATION
Zhang, Gu, Hurles, Lupski (2009) Ann Rev Genomics and Hum Genet 19:451-481
DNA replication model for genomic rearrangements
Fork Stalling
g and Template
p
Switching
g
FoSTeS
Mechanism
Jenny Lee
Clauda
Carvalho
Microhomology mediated joining by
priming DNA replication
Template driven juxtaposition of DNA
sequences separated
t d by
b large
l
genomic distances - template switch
1264
Lee , Carvalho, Lupski (2007) Cell 131:1235-1247
FoSTeS x 3
Cell 131:1235-1247, December 26, 2007
Jenny Lee
Claudia Carvalho
- Studied Pelizeaus-Merzbacher Dz
y y
g disorder
- CNS dysmyelinating
- ~ 70% due to different sized
(i.e. non-recurrent) PLP1 dup
join point
DNA replication mechanism:
Fork Stalling
Template Switching,
FoSTeS
1) Long distance template switching (120-550 Kb)
2) Tethered to original fork
3) Priming of DNA replication via microhomology
4) Template driven juxtaposition of discreet genomic
segments from different locations
MMBIR model
Hastings, Ira, Lupski (2009) PLoS Genetics 5
(Jan): e1000327
Hastings, Lupski, Rosenberg & Ira (2009)
Nat. Rev Genetics 10:551-64
collapsed replication fork
(one-ended, dsDNA
NOT DSB; i.e. two-ended dsDNA)
new low processivity fork
(disassociates repeatedly)
Reforms different template
Completes
p
replication
p
Breakpoint complexity
Phil Hastings
Microhomology at ‘join pt’ in FoSTeS/MMBIR: subtractive
R
E
P
L
I
C
A
T
I
V
E
ATGAATGACAGGATA...TCTAGACATATTCGA
Reference
ATGAATGACATATTCGA
Rearrangement Jct
Lee et al (2007) Cell 131:1235
Lee,
131:1235-1247
1247
Microhomology in the Shapiro model: additive
----ATGT
Transposable element
R f
Reference
Tn
ATCG----
+
TTCTAGGCACATTCTG
TTCTAGGCACA
Tn GCACATTCTG
Rearrangement Jct
Shapiro (1979) PNAS 76:1933-1937
Microhomology at sequenced breakpoint junctions in the human genome
Number of Number of
Number of rearrangements rearrangements Microhomology with with breakpoint length range*
breakpoints microhomology
sequenced
Reference
PLP1 (Xq22.2)
19
15 (79%)
2‐18
[1,2,3]
LIS1 (17p13.3)
Locus specific RAI1, PMP22 RAI1
PMP22
studies
(17p11.2p12)
6
6 (100%)
2‐27
[4]
36
26 (72%)
2‐33
[5,6,7,8]
STS (Xp22.31)
13
10 (77%)
2‐4
[9]
Vissers et al
Vissers et al.
38
29 (76%)
29 (76%)
2 30
2‐30
[10]
Genome‐wide Conrad et al.#
studies
Kidd et al.#
324
168 (52%)
2‐30
[11]
973
289 (30%)
2‐20
[12]
10871
7166 (66%)
2‐50
[13]
Mills et al.#
1. Lee et al., Cell 2007, 131:1235‐47. 2. Inoue et al., Am J Hum Genet 2002, 71:838‐853. 3. Woodward et al., J u Ge et 005, :966 98 . . Bi et al., Nat Genet 2009, 41:168‐177. 5.
et a ., at Ge et 009, : 68
. 5. Liu et al., Am J Hum Genet u et a .,
J u Ge et
Am J Hum Genet 2005, 77:966‐987. 4.
2011, 89:580‐588. 6. Zhang et al., Am J Hum Genet 2010, 86:462‐470. 7. Zhang et al., Nat Genet 2009, 41:849‐
853. 8. Zhang et al., Am J Hum Genet 2010, 86:892‐903. 9. Liu et al., Hum Mol Genet 2011, 20:1975‐1988. 10.
Vissers et al., Hum Mol Genet 2009, 18:3579‐3593. 11. Conrad et al.Nature Genetics 2010 42:386‐391 12.
Kidd et al., Cell 2010, 143:837‐847. 13. Mills et al., Nature 2011, 470:59‐65. FoSTeS/MMBIR favors gain (DUP, TRP, etc ) over loss of genomic material
etc.) over loss of genomic material
Pengfei
Liu
Replicative mechanism
important to evolution: i) gene duplication/triplication
ii) exon shuffling
Liu, et al. (2011) Am. J. Hum. Genet. 89: 580‐588 PLoS Genetics 5:1-9[e1000327] 2009
Microhomolgy:
-2 to 6 bp
-Alu - Alu
“One
One can experimentally
manipulate model
organisms to surmise
mechanism; however,
th relevance
the
l
to
t human
h
is by inference or
analogy alone – not by
p
direct experimental
observations.”
Zhang et al (2009)Trends
in Genetics 25: 298-397
Two types of triplication structures
yp
p
STS 3/61 = 4.9% of gains
de novo occurs by
double crossovers
Liu, et al (2011) HMG
type I triplication
reference
tandem
triplication
t
type
II triplication
t i li ti
reference
dup-inv/trp-dup
MECP2 13/58 = 22
22.4%
4% gains
Liu, Carvalho, Hastings, Lupski (2012) Current Opinions
in Genetics & Development 22: 211-220
a) arrayCGH findings – MECP2 locus in males with ID
+2
DUPp
Jct1
TRPp
p
J t2
Jct2
TRPd
DUPd
+1
aCGH
0
CEN
TEL
a
b
c
b) Actual complex rearrangement genomic structure
b) Actual complex rearrangement genomic structure
Carvalho, et al. (2011)
Nat Genet 43:1074-83
a
b
Jct1
c
Claudia
Carvalho
Jct2
b’
DUP‐TRP/INV‐DUP
a’
b’
c’
Carvalho
Claudia
a
Jct2
Strand
annealing and
extension
g
Jct1
b
c
d Strand invasion
at inverted ectopic
homology
e DNA
f Second fork
synthesis
collapse
a’
MMBIR b’
c’
a
Replication
a’
a’
a
b’
b
b’
b
b’
c’
c
c’
c
c’
d’
a’
b
b’
c’
c
d’
a
d’
b’
a
a
b’
b’
b
c
b’
or
d’
Fork
collapse
Carvalho, et al. (2011) Nature Genetics
43:1074-1082
b
c
a
b
c
d’
a
a’
h
a’
b’
c’
NHEJ
Ligation
DSB
a’
a
b’
b
c’
c
d’
b’
d’
Jct1
Jct2
Complex type II triplications
DUP
Claudia
Carvalho
Weimin Bi
Feng
Zhang
MECP2
LIS1
PMP22
TRP
DUP
Carvalho et al (2009)
Hum Mol Genet 18 :2188
:2188-2203
2203
Bi, et al (2009)
Nature Genetics 41 :168-177
Zhang et al (2009) Nature Genetics 41 :849-853
Mild Brain Structural Anomalies by MRI
duplication
triplication
B
A
LIS1 triplication
gross dysgenesis of the Corpus
callosum
marked cerebellar atrophy
duplication of YWHAE and LIS1
thinning corpus collosum splenium
mild cerebellar volume loss
Bi, et al (2009) Nature Genetics 41:168-177
Evolving new genes by DUP-TRP/INV-DUP
Inversion brings breakpoints into spatial proximity
perhaps within same replication factory
type II triplication
reference
dup-inv/trp-dup
AVPR2
TEX28
AVPR2
TEX28
TEX28/AVPR2
Jct1 Jct2
type II triplication evolves new genes by:
i) creating novel junctions
)
g
j
ii) inversion segment reading opposite strand
3
2
1
C*
Properties of MMBIR
replisome/polymerase
A
2X
tandem,
tandem
intra-chromosomal duplication
Original segment
3
Duplicated segment
2
132
C*
A
1
G*
A
*Lower Fidelity Primers
Ref_seq_1
Bkpt_jct
Ref seq 3
Ref_seq_3
Ref_seq_2
AGCAAGCTGGAATC
AGCAAGTCACGCTA
GTAAAGTCACGCCT
CGTATTGATGGCTA
Reduced
P
Processivity i i
FISH demonstrates an inverted
orientation of the middle copy in subjects S1–S6 with i
bj t S1 S6 ith
triplication (TRP) of subtelomere
All due respects
to Barbara:
It is NOT all BFB
genomic inversions: challenging to assay
BACKGROUND: Only two pathogenic inversions mediated by IR
BACKGROUND:
Only two pathogenic inversions mediated by IR
Two decades since the landmark Jane Gitschier study
A single inversion disrupting the factor VIII gene (F8) > 45% of patients with severe hemophilia A (MIM# 306700) !
IP‐LCRs can lead to abnormal disrupt a dosage‐sensitive gene(s) through NAHR
We delineated the genome‐wide distribution of IP‐ lCRs: 942 genes potentially disrupted!
DTIP‐LCRs > 1500 throughout genome: many potential genes can have dosage potentially changed Topics to be discussed:
1) Background – CNV & gene dosage
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &
other highly complex genomic changes
5) CNV & evolution, environmental mutagenesis
Cell (2011) 144:27‐40
Cancer implications of chromosome Cancer
implications of chromosome
catastrophe phenomena:
• 2‐3% of ALL cancer cell lines (N=746) • 25% of bone cancers (N=20)
25% f b
(N 20)
• Single catastrophic event NOT progressive rearrangement model (i.e. occurring sequentially and independently of one another over many cell cycles)
•Multiple cancer genes mutated in single mutational event – multigenic inheritance?
Liu et al. (2011)
146, 889-903.
10/12 pt referred for Developmental Delay (DD)
4/12 had Intellectual Disability + Behavioral Problems
12/12 had DD
http://www.bcm.edu/geneticlabs/
Subject BAB3105
Subject BAB3105
Multple regions of: dup, trp, and inv !
Note:
N
t None of the
N
f th
complexity is observed
in parents; consistent
in parents; consistent
with being generated
as part of a de novo p
event
Chromothripsis and Human Disease:
Piecing Together the Shattering Process
Ch i
Christopher A. Maher and Richard K. Wilson
h A M h
d Ri h d K Wil
Sanger –propose NHEJ
C ll (2012) 148: 29‐32
Cell
(2012) 148 29 32
Baylor propose FoSTeS/MMBIR
Figure 1. Chromothripsis Reshapes the Genomic Landscape in a Single Devastating Event
Three distinct types of highly complex genomic rearrangements Ch
Chromothripsis/
Chromoanasynthesis
Multiple de novo rearrangements: presented with
y
gp
peripheral
p
neuropathy
p y & developmental
p
delay
y
demyelinating
Location
Size
Type
Bkpt
features
Array
detection
180k
1
M
1p36
6.4 Mb
Duplication
1-bp micro
Y
Y
Pat
3q13q21
943 kb
Duplication
1-bp micro
Y
Y
Pat
3q29
104 kb
Duplication
Mosaic?
Y
5p12
440 kb
Duplication
7-bp micro
Y
Pat
5q33q34
5.8 Mb
Duplication
22q13 gain
Y
Y
Pat
1
9p13
1.2 Mb
Triplication
40-bp
complex;
10-bp insert
Y
Y
Mat,
Intra
2
17p11p12
6.0 Mb
Duplication
3-bp micro
Y
Y
Mat
22q11
48 kb
Duplication
NAHR？
Y
22q13
307 kb
Duplication
(inserted to
q q )
5q33q34)
3- and 48bp insert
Y
Genome-wide View
Pengfei Liu
Parent
of origin
1
2
Mat
A CNV mutator phenotype!
1
2
Multiple de novo rearrangements: presented with
demyelinating peripheral neuropathy & developmental delay
Occur on different parental chromosomes: Postzygotic event
Location
Size
Type
Bkpt
features
Array
detection
180k
1
M
Parent
of origin
1p36
6.4 Mb
Duplication
1-bp micro
Y
Y
Pat
3q13q21
943 kb
Duplication
1-bp micro
Y
Y
Pat
3q29
104 kb
Duplication
Mosaic?
Y
5p12
440 kb
Duplication
7-bp micro
Y
Pat
5q33q34
5.8 Mb
Duplication
22q13 gain
Y
Y
Pat
1
9p13
1.2 Mb
Triplication
40-bp
complex;
10-bp insert
Y
Y
Mat,
intra
2
17p11p12
6.0 Mb
Duplication
3-bp micro
Y
Y
Mat
22q11
48 kb
Duplication
NAHR？
Y
22q13
307 kb
Duplication
(inserted to
q q )
5q33q34)
3- and 48bp insert
Y
Genome-wide View
1
2
Mat
1
2
Multiple de novo rearrangements family #2
All occur on maternal chromosomes: germline event
Location
1
2
Size
Bkpt
features
Type
Array
blood
180k
1M
Array
Cell
Line
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Parent
of
origin
Mat
1p35.1p34.3
1.7 Mb
Duplication
1q21.2
491 kb
Triplication
3p21.1p14.3
4.2 Mb
Duplication
5q35.3
52 kb
Triplication
8q24.12q24.13
4.5 Mb
Duplication
Y
Y
10q24.33q25.1
4.7 Mb
Duplication
Y
Y
16p11.2
317 kb
Duplication
Y
Y
16q23.1q23.2
4.2 Mb
Duplication
Y
Y
Mat
16q24.3
310 kb
Duplication
Y
Y
Mat
19q13.2q13.32
4.3 Mb
Duplication
Y
Y
Mat
Xp11.23
211 kb
Duplication
Y
Mat
Y
NAHR？
Genome-wide View
Pengfei Liu
1
Mat
Mat
Mat
2
1
2
Topics to be discussed:
1) Background – CNV & gene dosage
2) CNV mechanisms - ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup
4) Chromothripsis vs chromoanasynthesis &
other highly complex genomic changes
5) CNV & evolution, environmental mutagenesis
What is the evolutionary rheostat !!!
CNV and Evolution
DNA REPLICATION MECHANISM
MECHANISM:
Fork Stalling Template Switching, FoSTeS/MMBIR
FoSTeS causes genomic dup and trip
p
rearrangements.
g
& complex
FoSTeS creates entirely novel genes by
g DNA DUP-TRP/INV-DUP.
inverting
FoSTeS may be a major mechanism
for duplication CNV and thus a major
driver of the Ohno “gene duplication /
divergence” evolutionary hypothesis.
divergence
FoSTeS may cause exon shuffling?
CNVs and evolution – inspired by the
Galapagos Islands (August 2008)
Intronic
enomic
Intrragenic Ge
(olligogenic?)
CNV enable rapid evolution of domesticated animals (
(Leif Andersson Lab, Uppsala SWEDEN)
, pp
)
Dorsal hair
ridge and predisposition to
d
dermoid
id sinus
i
133 kb dup (Contains FGF3, FGF4, FGF19,ORAOV1)
Rubin et al.2010
Nature 464:587-91
‘High growth’
19 kb del (3’ end of SH3RF2)
Premature hair
graying
g
y g and
susceptibility to
melanoma
Salmon Hillbertz
et al.2007
Nat Genet
39 1318 20
39:1318-20
4.6 kb dup (Intron 6 of STX17)
Pea-comb
Pea
comb
phenotype
WGS
Pielberg et al.2008
Nat Genet
40:1004-9
Wright et al. 2009
PLoS Genet 5:e1000512
Intergen
nic
~20-40X amplification (Intron 1 of SOX5)
Dark brown
plumage color
8.3 kb del (Upstream of SOX10)
Gunnarsson ett al.l
G
Pigment Cell
Melanoma Res
(In Press)
Reciprocal CNV, mirror traits and psychiatric dz
Crespi – evolution of the social brain
16p11.2 rearrangements diagnosed at Baylor MGL
16p11.2
rearrangements diagnosed at Baylor MGL
(http://www.bcm.edu/geneticlabs/ ) Mar 2008‐June 2012
Lupski (2012) Biological Psychiatry 72: 617-619
253 families provided
a molecular diagnosis!
CONCLUSIONS : CNV
What have we learnt?
1) NAHR favors del (2:1) whereas FoSTeS/MMBIR favors dup
2) Ectopic synapsis precedes ectopic crossing over/NAHR
p
can form de novo by
y double crossovers at LCR or from
3)) Triplications
pre-existing duplications
4) Triplications (type II) with non-recurrent breakpoints have a unique
p p
p
structure dup-trp/inv-dup
5) Telomeres may be particularly susceptible to replicative mutagenesis
6) CGR can form by MMBIR with template switches occurring at BOTH
homologous and micro-homologous
micro homologous substrate sequences; a ‘one
‘oneoff’ event, multiple genic changes - important for evolution!
7) CGR show many characteristics attributed to chromosome
catastrophe’s; the phenomena of chromothripsis described in 2-3%
2 3%
of all cancers: chromothripsis OR chromoanasynthesis or BOTH
mechanisms operative?
Issues relevant to
environmental mutagenesis
1)) CNV are important
p
for disease
(genomic disorders) & evolution
2) Do current mutagenesis assays (Ames test)
measure CNV formation?
3) C
Can we d
design
i such
h an assay?
?
4)) Are we introducing
g compounds
p
into our
environment that induce CNV mutagenesis?
5) What is the evolutionary ‘rheostat’
rheostat –
SNV (single nucleotide variation) or CNV
ACKNOWLEDEMENTS:
Gibbs Lab
& Baylor
+ Lupski Lab
&
http://imgen.bcm.tmc.edu/molgen/lupski/
Conclusion:
potential
t ti l CNV mutator
t t phenotype
h
t
1) A genome-wide
genome wide spectrum of de novo, large, rare
variant CNV
2) Brkpnt analyses reveal signatures [short
insertions flanked by microhomology + >SNV
rate 1000X ]of replicative mechanism (MMBIR)
3) M
Multiple
lti l d
de novo CNV ‘phenotype’
‘ h
t
’ can occur
post-zygotically or in germline (maternal)
Hypothesis: errors in cellular replication machinery
required
q
for MMBIR
Approach: WGS of entire trio (find smaller CNV)
ES to find gene responsible