genomic disorders, mechanisms for copy number variation ......genomic disorders: a new discipline of...
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Genomic disorders, mechanisms forcopy number variation & CNV in evolution
Exploring Human Genomic Plasticity & Environmental Stressors: Emerging
E id T l C
copy number variation, & CNV in evolution
Evidence on Telomeres, Copy Number Variation & Transposons
National Academies of Science
J R L ki M D Ph D D S
Washington, D.C4 October 2012
James R. Lupski, M.D., Ph.D., D.ScDepartment of Molecular & Human Genetics
& Department of PediatricsBaylor College of MedicineBaylor College of Medicine & Texas Children’s Hospital
Houston, TX
http://www.bcm.edu/geneticlabs/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:
1) Background – CNV & gene dosage( germ-line genomic variation )
) g g g
2) CNV mechanisms - ectopic synapsis (NAHR)2) CNV mechanisms ectopic synapsis (NAHR)
3) Triplications: dup - trp/inv – dup3) Triplications: dup trp/inv dup
4) Chromothripsis vs chromoanasynthesis &CGR4) Chromothripsis vs chromoanasynthesis &CGR(somatic intercellular variation)
5) CNV & evolution, environmental mutagenesis
CNV & phenotypes; an historical overview
Phenotypic Variation in DaturaDue to Changes inDue to Changes in
Chromosome Copy Number
The American NaturalistVol. 56 : 16-31, 1922
S i f i l
Dr. Albert Francis Blakeslee
Station for experimental evolution
Cold Spring Harbor L ICold Spring Harbor L.I., N.Y.
Calvin B. Bridges (1936) The Bar “Gene” a duplicationThe Bar Gene a 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 the total effect to the t e tota e ect to t egenic balance change [i.e. dosage]
and to theand to the position-effect change seems to be at present a matter
of taste”of taste - Calvin Bridges
Genic‐Scale Rearrangements & Human Di A Hi t i l P tiDisease: A Historical Perspective
α‐globin duplication
β‐thalassemia(mild)
Higgs, et al. (1980) Nature284:632‐5.
α‐globin duplication
Nathans, et al.Red‐green color blindness
Nathans, et al. (1986) Science232:203‐10
Glucocorticoid‐remediable ld t i (h t i )
Lifton, et al. (1992) Naturealdosteronism (hypertension) (1992) Nature355:262‐5
Genomic rearrangements? APP duplication d l t Al h i diand early‐onset Alzheimer disease
Delabar, et al. (1987) Science 235:1390‐2
APP Control Loci5
4
Quantification
e Do
sage
e Do
sage 3
2
Gen
eGen
2
1
0Southern to determine
d 0gene dosage
Two reports then argued against APPd l l h dduplication in Alzheimer disease
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
In 5 families with autosomal dominant early onset Alzheimer disease
Molecular mechanisms for chromosomal Molecular mechanisms for chromosomal syndromes, Mendelian dz and complex traits.syndromes, Mendelian dz and complex traits.
a) trisomy 21 b) copy number variation c) SNPs in promoter regions
A. Alzheimer disease
y , py , p
duplication of APPpromoters
APP coding exonsAPP SNPs
APP coding exons
B. Parkinson disease+ genic pt mut !
triplication of SNCA
a) copy number variation b) SNPs in promoter regions
promoters
duplication of SNCASNCA coding exons
SNPs
SNCA Shiga, Inoue, & Lupski (2007) Mendelian, non‐Mendelian,Multigenic inheritance and complex traits. In, The Molecular and Genetic Basis of Neurological and Psychiatric DiseaseRosenberg,, Prusiner, DiMauro, Barchi, (Eds.)
Human genome variation and disease:heuristic models to investigate genetic architecture of disease
>1 gene in CNVcontributes to phenotype?
C tiContiguousGene
Syndrome?
digenic &triallelictriallelic
Aneuploidy =a big CNV!
J.R. Lupski, J.W. Belmont, E. Boerwinkle, R.A. Gibbs (2011) Cell 147: 32‐43
a big CNV!
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 associatedhuman genome rearrangements
MEI – mobileelement insertion
MMBIR: microhomology-mediated, break inducedreplication
Feng Zhang
NAHR NHEJFoSTeS ×1 FoSTeS × 2
L1 RetrotranspositionFoSTeSp
OHP
TS
OH
TSDTSD
Zhang, Gu, Hurles, Lupski (2009) Ann Rev Genomics and Hum Genet 19:451-481
RECOMBINATION REPLICATION
The CMT1A duplication – a CNV paradigmRaeymakers, Timmerman, et al. (1991) Neuromuscular Disorders 1 :93-97
ProximalCMT1A-REP
DistalCMT1A-REP
y , , ( )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]
~ 70% of all CMT1 ptsCMT1A-REP CMT1A-REP
A B C D
PMP22
~ 70% of all CMT1 pts
76-90% of sporadic CMT1[de novo mutation]
A’ B’ C’ D’NORMAL: PMP22 = 2n[de novo mutation]
CMT1A: PMP22 = 3nHNPP: PMP22 = 1n
A B C B’ C’ D’ A’ D
CNV dzs:
CMT1A DUPLICATION HNPP DELETIONJCT JCTSchizophrenia
AutismObesity
Mechanism for deletion & i l d li tireciprocal duplication
NORMALNORMAL L. Potocki & J. R. LupskiNORMAL:NORMAL:““I I LIKE TO SWIM IN THELIKE TO SWIM IN THE OCEAN BUT I DO OCEAN BUT I DO NOTNOT LIKE TO SWIM IN THELIKE TO SWIM IN THE POOLPOOL ””
p
NOTNOT LIKE TO SWIM IN THELIKE TO SWIM IN THE POOLPOOL..
DELETION:DELETION:““II IKE TO SWIM IN THEIKE TO SWIM IN THE POOPOO ””““I I LIKE TO SWIM IN THELIKE TO SWIM IN THE POOLPOOL.”.”
DUPLICATIONDUPLICATIONDUPLICATION:DUPLICATION:““II LIKE TO SWIM IN THELIKE TO SWIM IN THE OCEAN BUT I DO NOTOCEAN BUT I DO NOT
LIKE TO SWIM IN THELIKE TO SWIM IN THE OCEAN BUT I DO NOTOCEAN BUT I DO NOTLIKE TO SWIM IN THELIKE TO SWIM IN THE OCEAN BUT I DO NOTOCEAN BUT I DO NOTLIKE TO SWIM IN THELIKE TO SWIM IN THE POOLPOOL.”.”
Genomic Disorders
i t NOTConcept predicated on two major premises:
- genomic rearrangements NOT sequence based changesq g
- genome architecture incites- 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 420disease traits Trends in Genetics 14:415-420
Lupski (2009) Genomic Disorders: ten years onGenome Medicine 1:42.1-42.11
Calvin B. Bridges (1936) The Bar “Gene” a duplicationThe Bar Gene a 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?
331
CMT1A duplication becomes a triplication in a family (unpublished)
BA
B33
B33
298
BA
B
severeCMT1A
mildCMT1A
BA
B33
2833
30B
AB
3
mildCMT1A Shay Ben-Shachar & Avi Orr-Urtreger; Tel Aviv
NAHR as the mechanism for recurrent genomic rearrangements
*A C
genomic rearrangements
*
*
interchromosome intrachromosome intrachromatid sister chromatid
deletion
duplication
inversion
Brecurrent translocation map
interchromosome intrachromosome(interchromatid)
intrachromatid sister chromatidexchange
B
d l ti d l ti d l tideletionduplication
deletionduplication
deletionisochromosome
iso17q –somaticisoY & isoX - constit.
Liu, et al. (2012) Curr Opin in Gen and Develop 22:211-220
Genomic disorders:a new discipline of medical geneticsa new discipline of medical genetics
Post-genomic eraBridges (1936)
Science 83:210‐211The Bar “Gene” duplicationcauses an eye phenotype
Lupski et al. (1991)Cell 66:219‐232
Genomic duplication causes neurological disease
Cheung et al. (2005)Genet. Med. 7:422‐432Clinical utilization of CGHcauses an eye phenotype neurological disease
1936 1991 2005
Feb ’04Clinical CMA
1998genomic disorders1936 1991 2005CMAdisordersdefined
Courtesy Dr Pengfei LiuN = 45,894;29FEB2012 rare dz day!N > 50,000 today!!!
Baylor Array CGH Team- clinically introduced highresolution human genome analyses Feb’04
Clinical DevelopmentClinical CytogeneticistsPatricia Hixson, Ph.D.
Sisi Bi, B.S.
Marcus Coyle B.S., M.A.
CheerleadersJim Lupski, M.D., Ph.D., D.Sc.
Art Beaudet, M.D.
Ankita Patel, Ph.D.
Sau wai Cheung, Ph.D.
Carlos Bacino, M.D.
Rodger Song, B.S.
Rebecca Davis, B.S.
Lu Yang, B.S.
General ManagerSean Kim, M.B.A.
Pawel Stankiewicz, M.D., Ph.D.
Seema Lalani, M.D.
Weimin Bi, Ph.D.
Genetic Counselors
Amanda Fullerton, B.S.
AdministrationJeff Mize, M.B.A., C.P.A.
Robert Johnson, Ph.D.
Mitochondrial Disease Arrays
Amy Breman, PhD.
Patricia Ward, M.S.
Sandra Peacock, M.S.
Alicia Braxton, M.S. Array DevelopmentMarketingMike Frazier, B.S.
ArraysLee-Jun Wong, Ph.D.
Laura Ellis, M.SStatistics/BioinformaticsChad Shaw, Ph.D.
Pawel Stankiewicz, M.D., Ph.D.
Tomek Gambin, B.S.
Prenatal Genetics
T. Brandon Perthuis, B.S.
Aloma Geer, Ph.D.
Eric Burgess, B.S.
CAleksandar Milosavljevic, Ph.D.
Jian Li, B.S. Christine Eng, M.D.
Ignatia Van Den Veyver, M.D.
.CMA - chromosomalmicroarray analyses
N = 50,310 (19 Aug 2012)
Genome‐wide CNV studies in patients:lessons learntlessons learnt
A locus can be subject to recurrent or
Apparently Simple
A locus can be subject to recurrent ornon‐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.
6 4New mutations are quite frequent for CNV (10‐6 to 10‐4) compared to SNV (10‐8) [100X – 10,000X !!!].
Potocki-Lupski syndrome (PTLS;MIM #610883)
2000, seven patients with common duplication were described;
2007 ltidi i li li i l t d2007, multidisciplinary clinical study
Potocki et al (2000) Nat Genet 24:84-87Potocki et al (2007) AJHG 80:633-649
Definition of a genomic disorder – from mechanism to clinical delineation
PTLS Family Conference July 2012 Texas Children’s Hospital Houston,TXp ,
smile – say cheese!
silly face! > 300 patients with PTLS in families’ database
Rearrangement frequency at 17p11.2
Feng Zhang
FAMILIESSTUDIED:
PTLS d li tiPTLS duplicationN=79
SMS deletionN=131
Pengfei Liu
Distribution of different mechanisms in del/dup Recurrent Nonrecurrent simple Complex (FoSTeS or Total(NAHR) (FoSTeS or NHEJ) 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 identifiedii) 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 17p11.2all three LCRs are similar in identity; ~98.6% !
What makes one recurrent rearrangement more prevalent than another?What makes one recurrent rearrangement more prevalent than another?i.e. what determines the NAHR frequency at a locus?
LCR
Inter-LCR distance
LCRlength
NAHR mediated rearrangement frequency at a given locus correlates positively with LCR length & inversely influenced by
inter‐LCR distancesinter‐LCR distances
LCR LengthLCR DistanceLCR Length (Kb)
LCR Length
log (Fre
ln (Freq
LCR distanceequency)
quency)
CommonRecurrent
UncommonRecurrent 1
UncommonRecurrent 2
Red: DupGreen: Del
Legend: NAHR
SynapsisSynapsis‐‐ Alignment of homologues in meiosisTerry 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 ofectopic crossing over ?
PengfeiLiu *
ectopic crossing over ?
Liu, et al. (2011) Am J Hum Genet 89:580-588* 2012 Cotterman Award
c/w: i) NAHR & AHR hotspots coincideii) same PRDM9 hotspot motif usediii) yst synaptonemal complex mutants abolish ectopic HR!!! Shinohara Lab
Depletion of Zip1 an essential component of yeast synaptonemal
Evidence in yeast
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 associatredhuman genomic rearrangements
MEI – mobileelement insertion
MMBIR: microhomology-mediated, break inducedreplication
Feng Zhang
NAHR NHEJFoSTeS ×1 FoSTeS × 2
L1 RetrotranspositionFoSTeSp
OHP
TS
OH
TSDTSD
Zhang, Gu, Hurles, Lupski (2009) Ann Rev Genomics and Hum Genet 19:451-481
RECOMBINATION REPLICATION
DNA replication model for genomic rearrangementsFork Stalling and Template Switchingg p g
FoSTeS
Mechanism
Jenny LeeClauda Carvalho
Microhomology mediated joining bypriming DNA replication
Template driven juxtaposition of DNAt d b lsequences separated by large
genomic distances - template switch
FoSTeS x 3 1264
Lee , Carvalho, Lupski (2007) Cell 131:1235-1247
Cell 131:1235-1247, December 26, 2007 Jenny Lee
Claudia Carvalho
- Studied Pelizeaus-Merzbacher Dz- CNS dysmyelinating disorder
DNA replication mechanism:
y y g- ~ 70% due to different sized
(i.e. non-recurrent) PLP1 dup
Fork StallingTemplate Switching,
FoSTeSjoin point
1) Long distance template switching (120-550 Kb)2) Tethered to original fork3) Priming of DNA replication via microhomology3) Priming of DNA replication via microhomology4) Template driven juxtaposition of discreet genomic
segments from different locations
MMBIR modelHastings, Ira, Lupski (2009) PLoS Genetics 5
collapsed replication fork
(Jan): e1000327Hastings, Lupski, Rosenberg & Ira (2009)
Nat. Rev Genetics 10:551-64collapsed replication fork(one-ended, dsDNANOT DSB; i.e. two-ended dsDNA)
new low processivity fork(disassociates repeatedly)
Reforms different template
Completes replicationp p
Breakpoint complexity Phil Hastings
Microhomology at ‘join pt’ in FoSTeS/MMBIR: subtractive
ATGAATGACAGGATA...TCTAGACATATTCGA
Reference
REP
ATGAATGACATATTCGA
Rearrangement JctLee et al (2007) Cell 131:1235 1247
PLI
Microhomology in the Shapiro model: additive
Lee, et al (2007) Cell 131:1235-1247I CA
Tn----ATGT ATCG----
TTCTAGGCACATTCTGR f
Transposable element +TI
TTCTAGGCACATTCTG
TTCTAGGCACA GCACATTCTGTn
ReferenceVE
Rearrangement JctShapiro (1979) PNAS 76:1933-1937
Microhomology at sequenced breakpoint junctions in the human genome
Number of Number ofrearrangements with
breakpoints sequenced
Number of rearrangements with breakpoint microhomology
Microhomology length range* Reference
Locus specific studies
PLP1 (Xq22.2) 19 15 (79%) 2‐18 [1,2,3]
LIS1 (17p13.3) 6 6 (100%) 2‐27 [4]
RAI1 PMP22studies RAI1, PMP22 (17p11.2p12) 36 26 (72%) 2‐33 [5,6,7,8]
STS (Xp22.31) 13 10 (77%) 2‐4 [9]
Vissers et al 38 29 (76%) 2 30 [10]
Genome‐wide studies
Vissers et al. 38 29 (76%) 2‐30 [10]
Conrad et al.# 324 168 (52%) 2‐30 [11]
Kidd et al.# 973 289 (30%) 2‐20 [12]
Mills et al.# 10871 7166 (66%) 2‐50 [13]
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., Am J Hum Genet 2005, 77:966‐987. 4. Bi et al., Nat Genet 2009, 41:168‐177. 5. Liu et al., Am J Hum Genet J u Ge et 005, :966 98 . . et a ., at Ge et 009, : 68 . 5. u et a ., J u Ge et2011, 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 materialetc.) over loss of genomic material
PengfeiLiu
Replicative mechanism important to evolution: i) gene duplication/triplication
Liu, et al. (2011) Am. J. Hum. Genet. 89: 580‐588
important to evolution: i) gene duplication/triplicationii) exon shuffling
PLoS Genetics 5:1-9[e1000327] 2009
Microhomolgy:Microhomolgy:Microhomolgy:-2 to 6 bp-Alu - Alu
Microhomolgy:-2 to 6 bp-Alu - Alu
“One can experimentallyOne can experimentally manipulate model organisms to surmisemechanism; however, th l t hthe relevance to humanis by inference oranalogy alone – not bydirect experimentalpobservations.” Zhang et al (2009)Trendsin Genetics 25: 298-397
Two types of triplication structuresyp pSTS 3/61 = 4.9% of gainsde novo occurs bytype I triplication
reference
tandem
double crossoversLiu, et al (2011) HMG
triplication
t II t i li ti
reference
type II triplication
dup-inv/trp-dup
MECP2 13/58 = 22 4% gainsMECP2 13/58 = 22.4% gains
Liu, Carvalho, Hastings, Lupski (2012) Current Opinions in Genetics & Development 22: 211-220
a) arrayCGH findings – MECP2 locus in males with ID
+2 TRPp TRPdJct1
J t2
+1
+2
aCGHDUPp
pDUPdJct2
0
CEN TEL
c
b) Actual complex rearrangement genomic structure
a b
b) Actual complex rearrangement genomic structureJct1 Jct2
ClaudiaCarvalhoCarvalho, et al. (2011)
Nat Genet 43:1074-83
a b a’ b’b’c c’
DUP‐TRP/INV‐DUP
Jct2aCarvalhoClaudia
Jct1
Strandannealing and
extension
g
b
a
b’
a’
Strand invasionat inverted ectopic
homology
c d DNAsynthesis
eh
b f
b
a
b’
a’
Second forkcollapse
b’ b’
ba
a’
a
b
a
bb’cc’
MMBIR
b
aa’
b
aa’
bb’cc’
d’ d’
b’ b’
Jct1Replication cb’a’b’
b’ b
c’ c
d’or
bb’cc’
d’
bb’cc’
b’
d’ Jct2
c’d’DSB
b’a’
ba
NHEJLigation
Fork collapse
b’c’d’
c
Carvalho, et al. (2011) Nature Genetics43:1074-1082
Complex type II triplicationsDUP TRP DUPDUP TRP DUP
MECP2 ClaudiaCarvalho Carvalho et al (2009)
Hum Mol Genet 18 :2188-2203Hum Mol Genet 18 :2188 2203
LIS1Weimin Bi Bi, et al (2009) Nature Genetics 41 :168-177
PMP22Feng Zhang Zhang et al (2009) Nature Genetics 41 :849-853
Mild Brain Structural Anomalies by MRI
A B
triplication duplication
LIS1 triplication
gross dysgenesis of the Corpus callosum
duplication of YWHAE and LIS1
thinning corpus collosum spleniummild cerebellar volume losscallosum
marked cerebellar atrophymild cerebellar volume loss
Bi, et al (2009) Nature Genetics 41:168-177
Evolving new genes by DUP-TRP/INV-DUPInversion brings breakpoints into spatial proximity
perhaps within same replication factory
reference
type II triplicationAVPR2 TEX28
dup-inv/trp-dupTEX28/AVPR2AVPR2 TEX28
Jct1 Jct2
type II triplication evolves new genes by:i) creating novel junctions) g j
ii) inversion segment reading opposite strand
123Properties of MMBIRreplisome/polymerase
tandem2X
AC*
tandem,intra-chromosomal duplication
Original segment Duplicated segmentOriginal segment Duplicated segment
A G AC *
1322 1
*
3
A G AC*Lower Fidelity
AGCAAGCTGGAATCAGCAAGTCACGCTAGTAAAGTCACGCCT
ReducedP i i
Ref_seq_1Bkpt_jctRef seq 3 GTAAAGTCACGCCT
CGTATTGATGGCTAProcessivity Ref_seq_3
Ref_seq_2Primers
FISH demonstrates an invertedorientation of the middle
i bj t S1 S6 ithcopy in subjects S1–S6 with triplication (TRP) of subtelomere
All due respects to Barbara:to Barbara:
It is NOT all BFB
BACKGROUND: Only two pathogenic inversions mediated by IR
genomic inversions: challenging to assay
BACKGROUND: Only two pathogenic inversions mediated by IRTwo decades since the landmark Jane Gitschier studyA 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 chromosomeCancer implications of chromosome catastrophe phenomena:
• 2‐3% of ALL cancer cell lines (N=746)
25% f b (N 20)• 25% of bone cancers (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)10/12 pt referred for Developmental Delay (DD)4/12 had Intellectual Disability + Behavioral Problems12/12 had DD
http://www.bcm.edu/geneticlabs/http://www.bcm.edu/geneticlabs/
Subject BAB3105Subject BAB3105
Multple regions of: dup, trp, and inv !
N t N f thNote: None of thecomplexity is observedin parents; consistentin parents; consistentwith being generatedas part of a de novo pevent
Chromothripsis and Human Disease:Piecing Together the Shattering ProcessCh i h A M h d Ri h d K Wil C ll (2012) 148 29 32Christopher A. Maher and Richard K. Wilson Cell (2012) 148: 29‐32
Sanger –propose NHEJ Baylor propose FoSTeS/MMBIR
Figure 1. Chromothripsis Reshapes the Genomic Landscape in a Single Devastating Event
Three distinct types of highly complex genomic rearrangements
ChChromothripsis/Chromoanasynthesis
Multiple de novo rearrangements: presented with demyelinating peripheral neuropathy & developmental delay
Location Size Type Bkptfeatures
Arraydetection Parent
of origin180k 1
y g p p p y p y
Pengfei Liu180k 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? Y15p12 440 kb Duplication 7-bp micro Y Pat
5q33q34 5.8 Mb Duplication 22q13 gain Y Y Pat
9p13 1.2 Mb Triplication40-bp
complex;10-bp insert
Y Y Mat,Intra
1
1
17p11p12 6.0 Mb Duplication 3-bp micro Y Y Mat
22q11 48 kb Duplication NAHR? Y
22q13 307 kbDuplication(inserted to 5q33q34)
3- and 48-bp insert Y Mat
2 2
A CNV mutator phenotype!q q )
Genome-wide View 1 2
A CNV mutator phenotype!
Multiple de novo rearrangements: presented with demyelinating peripheral neuropathy & developmental delay
Location Size Type Bkptfeatures
Arraydetection Parent
of origin180k 1
Occur on different parental chromosomes: Postzygotic event
1
180k 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? Y15p12 440 kb Duplication 7-bp micro Y Pat
5q33q34 5.8 Mb Duplication 22q13 gain Y Y Pat
9p13 1.2 Mb Triplication40-bp
complex;10-bp insert
Y Y Mat,intra
1
217p11p12 6.0 Mb Duplication 3-bp micro Y Y Mat
22q11 48 kb Duplication NAHR? Y
22q13 307 kbDuplication(inserted to 5q33q34)
3- and 48-bp insert Y Mat
2
Genome-wide View 1 2
q q )
Multiple de novo rearrangements family #2All occur on maternal chromosomes: germline event
Location Size Type Bkptfeatures
Arrayblood Array
Cell Line
Parent of
origin180k 1M
Pengfei Liu
1p35.1p34.3 1.7 Mb Duplication Y Y Y Mat
1q21.2 491 kb Triplication Y Y
3p21.1p14.3 4.2 Mb Duplication Y Y Y Mat
5q35.3 52 kb Triplication NAHR? Y Y
1
8q24.12q24.13 4.5 Mb Duplication Y Y Y Mat
10q24.33q25.1 4.7 Mb Duplication Y Y Mat
16p11.2 317 kb Duplication Y Y
16q23.1q23.2 4.2 Mb Duplication Y Y Mat
1
2 216q24.3 310 kb Duplication Y Y Mat
19q13.2q13.32 4.3 Mb Duplication Y Y Mat
Xp11.23 211 kb Duplication Y Mat
Genome-wide View 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 mutagenesisWhat is the evolutionary rheostat !!!
CNV and EvolutionDNA REPLICATION MECHANISMDNA REPLICATION MECHANISM: Fork Stalling Template Switching, FoSTeS/MMBIR
FoSTeScauses genomic dup and trip& complex rearrangements.p g
FoSTeS creates entirely novel genes byinverting DNA DUP-TRP/INV-DUP.
FoSTeS may be a major mechanismfor duplication CNV and thus a major
g
for duplication CNV and thus a major driver of the Ohno “gene duplication / divergence” evolutionary hypothesis.divergence evolutionary hypothesis.
FoSTeS may cause exon shuffling?
CNVs and evolution – inspired by the Galapagos Islands (August 2008)Galapagos Islands (August 2008)
CNV enable rapid evolution of domesticated animals (Leif Andersson Lab, Uppsala SWEDEN)( , pp )
enom
iclig
ogen
ic?)
Dorsal hair ridge and pre-disposition to d id i
Salmon Hillbertz et al.2007Nat Genet
39 1318 20Ge
(ol dermoid sinus 39:1318-20133 kb dup (Contains FGF3, FGF4, FGF19,ORAOV1)
‘High growth’ Rubin et al.2010Nature 464:587-91ra
geni
c
Pielberg et al.2008
Premature hair graying and
19 kb del (3’ end of SH3RF2)Intr WGS
Pea comb
4.6 kb dup (Intron 6 of STX17)
Pielberg et al.2008Nat Genet 40:1004-9
g y gsusceptibility to melanoma
Intr
onic
G t l
Pea-comb phenotype
Wright et al. 2009PLoS Genet 5:e1000512
nic
~20-40X amplification (Intron 1 of SOX5)
Gunnarsson et al.Pigment Cell
Melanoma Res (In Press)
Dark brown plumage color
Inte
rgen
8.3 kb del (Upstream of SOX10)
Reciprocal CNV, mirror traits and psychiatric dzCrespi – evolution of the social brainCrespi – evolution of the social brain
16p11.2 rearrangements diagnosed at Baylor MGL16p11.2 rearrangements diagnosed at Baylor MGL(http://www.bcm.edu/geneticlabs/ ) Mar 2008‐June 2012
253 families provided a molecular diagnosis!Lupski (2012) Biological Psychiatry 72: 617-619
CONCLUSIONS : CNVWhat have we learnt?
1) NAHR favors del (2:1) whereas FoSTeS/MMBIR favors dup1) NAHR favors del (2:1) whereas FoSTeS/MMBIR favors dup2) Ectopic synapsis precedes ectopic crossing over/NAHR 3) Triplications can form de novo by double crossovers at LCR or from ) p y
pre-existing duplications4) Triplications (type II) with non-recurrent breakpoints have a unique
structure dup-trp/inv-dupp p p5) Telomeres may be particularly susceptible to replicative mutagenesis6) CGR can form by MMBIR with template switches occurring at BOTH
homologous and micro homologous substrate sequences; a ‘onehomologous and micro-homologous substrate sequences; a ‘one-off’ 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%catastrophe’s; the phenomena of chromothripsis described in 2-3% of all cancers: chromothripsis OR chromoanasynthesis or BOTH mechanisms operative?
Issues relevant to environmental mutagenesisenvironmental mutagenesis
1) CNV are important for disease ) p(genomic disorders) & evolution
2) Do current mutagenesis assays (Ames test)2) Do current mutagenesis assays (Ames test)measure CNV formation?
3) C d i h ?3) Can we design such an assay?
4) Are we introducing compounds into our) g penvironment that induce CNV mutagenesis?
5) What is the evolutionary ‘rheostat’ –5) What is the evolutionary rheostat –SNV (single nucleotide variation) or CNV
ACKNOWLEDEMENTS: Gibbs Lab& Baylor& Baylor
+ Lupski Lab&
http://imgen.bcm.tmc.edu/molgen/lupski/
Conclusion: t ti l CNV t t h tpotential CNV mutator phenotype
1) A genome-wide spectrum of de novo, large, rare1) A 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 lti l d CNV ‘ h t ’3) Multiple de novo CNV ‘phenotype’ can occur post-zygotically or in germline (maternal)
Hypothesis: errors in cellular replication machineryrequired for MMBIRq
Approach: WGS of entire trio (find smaller CNV)ES to find gene responsible