Genomic disorders, mechanisms for copy number variation ......Genomic disorders: a new discipline of medical genetics Post-genomic era Bridges (1936) Science 83:210‐211 The Bar “Gene”

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)


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:






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)







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







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


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


q q )

Multiple de novo rearrangements family #2All occur on maternal chromosomes: germline event


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


1

2 216q24.3 310 kb Duplication Y Y Mat


Xp11.23 211 kb Duplication Y Mat







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

Documents

Genomic disorders, mechanisms for copy number variation ......Genomic disorders: a new discipline of medical genetics Post-genomic era Bridges (1936) Science 83:210‐211 The Bar “Gene”