“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

1The screen versions of these slides have full details of copyright and acknowledgements

“Genomic Disorders: Mechanisms for Copy-Number Variation

(CNV) and Clinical Implementation of High-Resolution Genome Analysis”

James R. Lupski, M.D., Ph.D.D t t f M l l & H G ti

1

Department of Molecular & Human GeneticsDepartment of PediatricsBaylor College of Medicineand Texas Children’s HospitalHouston, TX

http://www.bcm.edu/geneticlabs/

Structural variation and disease

• Clinical array rationale and design

– Targeted genomic regions

– Coalescence to whole genome coverage

• Genomic rearrangement mechanisms

2

– Recombination: NAHR, NHEJ

– Replication: FoSTeS

• De novo rearrangements and birth defects in neonates

• Aneuploidy, Mendelian disease, complex traits: a continuum

Replication mechanism for CNVFork Stalling and Template Switching

FoSTeS

3dup PLP1

Pelizaeus-Merzbacher[PMD; MIM 312080]

dup MECP2Lubs XLMR

[MRXS; MIM 300262]

dup RAI1Potocki-Lupski

[PTLS; MIM 610883]

Jenny Lee Claudia Carvalho Feng Zhang

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

2The screen versions of these slides have full details of copyright and acknowledgements

Baylor college of medicine array CGH teamClinical cytogeneticists

Sau Wai Cheung, Ph.D.

Ankita Patel, Ph.D.

Carlos Bacino, M.D.

Sue Kang, Ph.D.

Sahoo Trilochan, M.D.

Pawel Stankiewicz, M.D., Ph.D.

Seema Lalani, M.D.

Array development

A. Craig Chinault, Ph.D.

Clinical development

Lisa White, Ph.D.

Susan Venable, B.A.

Cheerleaders

Jim Lupski, M.D., Ph.D.

Art Beaudet, M.D.

Administration

Jeff Mize, M.B.A., C.P.A.

General manager

Robert Johnson, Ph.D.

Mitochondrial disease arrays

Lee-Jun Wong, Ph.D.

4

,

Xinyan Liu, M.D.

Genetic counselors

Patricia Ward, M.S.

Amber Pursley, M.S.

Claudia Soler, M.D.

Statistics/bioinformatics

Chad Shaw, Ph.D.

Aleksandar Milosavljevic, Ph.D.

Jian Li, B.S.

Zhishuo Ou, M.D.

Pawel Stankiewicz, M.D., Ph.D.

Svetlana Yatsenko, M.D.

Prenatal genetics

Christine Eng, M.D.

Ignatia Van Den Veyver, M.D.

Marketing

Mike Frazier, B.S.

T. Brandon Perthuis, B.S.

Alejandra Hamilton Quick, B.S.

Detection of clinically relevant CNV

BAC V4 Feb, 04

BAC V5 July, 05

BAC V6 Nov, 06

OLIGO V6 April, 07

Interrogating probes

366 BACs

853 BACs

1476 BACs

44,000 Oligos

5

*Final percentage will increase as parental studies distinguish de novofrom inherited abnormalities; (Prepared May 28, 2007)

Abnormal diagnoses

62 406 243 70

CMA index cases 955 4492 1923 544

Detection rate 6.50% 9.04% ~12% ~12.8%*

Chromosomal microarray analysis (CMA)

6

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

3The screen versions of these slides have full details of copyright and acknowledgements

Design guideline

Evenly distribution BAC-emulated targeting

V7

V7.1

7

Targeted regions:• Selected genes (424)• Known disease regions (554)• Predicted unstable regions

between LCRs (295)• V6.4 backbone BACs (912)

Excluding regions:• Repetitive elements• Low copy repeats• Assembly gaps• Copy number polymorphism

(TCAG V1+UCSC)

3X 2X X

Design exampleGene (NIPBL) 3X Disease regions

(Cornelia de Lange) 2X

8Backbone regions X

Structural variation and disease

Clinical array rationale and design

• Targeted genomic regions

• Coalescence to whole genome coverage

Genomic rearrangement mechanisms

9

Genomic rearrangement mechanisms

• Recombination: NAHR, NHEJ

• Replication: FoSTeS

De novo rearrangements and birth defects in neonates

Aneuploidy, Mendelian disease, complex traits: a continuum

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

4The screen versions of these slides have full details of copyright and acknowledgements

- Genomic rearrangements not sequence based changes

Genomic disorders

10

- Genome architecture incites genome instability

Lupski (1998) Trends in Genetics 14: 415-420

11

– Gene

– Low-copy repeat (LCR)

– Complex genomic architectural elements (e.g. palindromes or cruciforms)

– Smallest region of overlapSRO

Recurrent rearrangements in proximal 17p

CMT1A

HNPP

~ frequency observed

> 99%

> 99%

TEL

i(17q)

RAI1

12

70-80%

4-5%

PTLSCMT1A-REP

SMS-REP

LCR17pA

SMS

SMS

~ 70%

DeletionDuplication

PMP22

Lupski and Stankiewicz (2005) PLOS Genet. 1(6):e49

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

5The screen versions of these slides have full details of copyright and acknowledgements

Breakpoint mapping in proximal 17p~ 4 Mb

~ 5 Mb

Common recurrent

Uncommon recurrent

Uncommon nonrecurrent

Smith-Magenis deletion rearrangements

13

Interstitial deletions

~ 4 MbCommon recurrent

Uncommon nonrecurrent

Interstitial duplications

Potocki-Lupski duplication rearrangements

Poly-Ser

RAI1 point mutations identified in SMS patients without deletion

Frameshift Nonsense Missense

III III

NLSIV V VI

PHD

Poly-Gln

NLSPoly-SerATG TAG

(Gln)9-18

14

(1) Slager et al., (2003) Nat Genet 33: 466-468

(2) Bi et al., (2004) Hum Genet 115: 515-524

(3) Girirajan et al., (2005) J Med Genet 42: 820-826

(4) Bi et al., (2006) Am J Med Genet 140: 2454-2463

Polymorphic CAG repeat length associated with:– Response to neuroleptics in schizophrenia

patients[Joober et al., (1999) Am J Hum Genet 88: 694-699]

– Age at onset variability in spinocerebellar ataxia (SCA2)[Hayes et al., (2000) Hum Mol Genet 9: 1753-1758]

CCCCCCC

NAHR NHEJ FoSTeS

Mechanisms for genomic rearrangements associated with genomic disorders

15Gu, Zhang, and Lupski (2008) Pathogenetics 1:4

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

6The screen versions of these slides have full details of copyright and acknowledgements

a

Non allelic homologous recombination

16

Interchromosomal IntrachromatidInterchromatid

b

Rearrangements:

Outcome: dup del dup deldel

• Homologous recombination – Homologous sequences (LCR/SD) as substrates – Recombination hotspots

Non allelic homologous recombination NAHR

17Stankiewicz and Lupski (2002) Trends in Genetics 18: 74-82

– Recombination hotspots– Gene conversion

• Deletion/duplication, inversion– Direct vs. inverted repeats

Lessons learned from NAHR

1. Genome architecture is important to genome instability (repeat size, % identity, distance between repeats, dir vs. inv repeats)

2. Reciprocal rearrangements (i.e. duplication/deletion),

18

p g ( p )but not equivalent

3. Recombination hotspots (NAHR hotspots); Can coincide with AHR hotspots

4. Can predict genomic regions that can undergo duplication/deletion

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

7The screen versions of these slides have full details of copyright and acknowledgements

Reciprocalrecombinations

1) Charcot-Marie-Tooth disease type 1A/ hereditary neuropathy with pressure palsies

2) Smith-Magenis syndrome/dup(17)(p11.2p11.2) syndrome

19

Recombinant products

7.3 Kb 6.9 Kb 14.5 Kb1.7 Kb

Bi et al., (2003)AJHG

73:1302-15

Reiter et al., (1996)Nat Genet

12: 288-297

DNA replication mechanism: fork stalling template switching

Cell 131: 1235-1247, December 26, 2007

20join point

DNA replication model for genomic rearrangements

Fork Stalling and Template Switching

FoSTeSMechanism

– Microhomology mediated joining– Template driven juxtaposition of DNA

21FoSTeS x 31264

Lee et al., (2007) Cell 131: 1235-1247

p j psequences separated by large genomic distances

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

8The screen versions of these slides have full details of copyright and acknowledgements

Proposed FoSTeS model for genomic

rearrangements causing genomic

disordersLee et al., (2007) Cell 131:1235-1247

B

C

22

, ( )

D

E

– Long distance template switching between forks (120Kb – 550Kb leaps)

– Tethered to original fork– Priming of DNA replication

via microhomology

Complex genomic architecture where fork stalls and templates switches

23Lee et al., (2007) Cell 131:1235-1247

24NHEJ

Lee et al., (2007) Cell 131:1235-1247

NHEJNHEJ or FoSTeSx1

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

9The screen versions of these slides have full details of copyright and acknowledgements

MECP2 triplication

25

Clinical (>80%; N=53):– Males, DD, Sz– Infantile hypotonia– Absent speech– Recurrent infections

26

• Submicroscopic imbalances in subtelomeric regions [N = 5,380]

• Red bars represent loss (deletion) whereas green bars represent gain (duplication)

• Abnormal 4.4%; The most frequently identified rearrangements map to 1p, 22q and Xq

• 10 Mb coverage, 41 subtelomeric regions [~ 1/10 of genome, but 1/3 of all abnormalities detected]

Five out of 26 (23%) patients with MECP2 duplications had presented complex

rearrangements

27

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

10The screen versions of these slides have full details of copyright and acknowledgements

Complex MECP2 duplications

2622

2624

5/26 (19%) nonrecurrent MECP2 duplications

are complex

28

MECP2_5

JV

2681

Breakpoint junctions group near or at the segmental duplications

around MECP2

29

Low copy repeats (LCRs) around MECP2 gene

30Opsin array: frequent rearrangements and gene conversions produce

common variation in color vision and red-green color deficient subjects

Del Gaudio et al., 2006

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

11The screen versions of these slides have full details of copyright and acknowledgements

Low copy repeats (LCRs) around MECP2 gene (2)

31

Polymorphic inversion present in 33%of females of European descent

~99% similarityDel Gaudio et al., 2006

Potocki-Lupski syndrome (PTLS;MIM #610883)

32• 2000, seven patients with common

duplication were described• 2007, multidisciplinary clinical study

Potocki et al., (2000) Nat Genet 24: 84-87Potocki et al., (2007) AJHG 80: 633-649

First predicted microduplication syndrome from reciprocal recombination

NAHR mechanism

33

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

12The screen versions of these slides have full details of copyright and acknowledgements

dup(17)(p11.2p11.2) syndrome

• Less severe than del17p11.2• Major features

– MR/DD, hypotonia– Craniofacial features

Triangular face

34

g– Neurobehavioral abnormalities

HyperactivityAttention deficitAutistic features

– Dental abnormalities

Potocki et al., (2007) Am J Hum Genet. 80: 633-649

Feng Zhang

Complex 17p duplications

9/14 (64.3%) complex;8 by array and 1 of remaining 6 the complexity only visualized

by brkpt jct sequence

2543

621

35Duplication Deletion Triplication

2543

2337

2211

1458

1229

2695

2711

PMP22 CNV detected by abnormal MLPA for CMT1A duplication

year dup/del test nml dup del

2007 4261 3472 549 194

36

• MLPA unusual in 7 samples

• Frequency of detecting dup/del = (549+194)/4261 = 17.5%

• Frequency of unusual MLPA = 7/(549+194) = 0.8%

• Estimated NAHR at CMT1A/HNPP locus = 99.2%

2007 4261 3472 549 194

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

13The screen versions of these slides have full details of copyright and acknowledgements

Nonrecurrent genomic rearrangementsinvolving PMP22 include complex structures

37

Junction analysis of A15 deletion in PMP22

Dist_Ref2+ TCGTAAAAGGTGCCCAACCTCACTAGCAACCAAGGAAATGCAAGAGAAACCCATGAGGAGGGTGACACCAA15_23+ TCGTAAAAGGTGCCCAACCTATAGCCATCTATTCCTTGACAAGGTGCCCAACCTCACTAGCAACCAAGGAProx_Ref23- CATTCTTATTTTCAGAACCTATAGCCATCTATTCCTTGACAAGATCTGTTGGGTTGGGTTGCACTAGACTDist_Ref3+ TTAGCAAAGGAGAAATATGAACAGCCAATAAACATCGTAAAAGGTGCCCAACCTCACTAGCAACCAAGGA

Dist_Ref1+ TTGATGTTTTCCAGTCTAGTGCAACCCAACCCAACAGATCTTGTCAAGGAATAGATGGCTATAGGTTCTGA15_1+ TTGATGTTTTCCAGTCTAGTGCAACCCAACCCAACAAAGAGAAAACAGCTAAGTATAAAATTGAAAAGCCProx_Ref1+ TCGCATCATTAACAAAATTAAATTACAGACAGAACAAAGAGAAAACAGCTAAGTATAAAATTGAAAAGCC

Junction 1

Junction 2 and 3

AACAAACA (98 bp)

38

AACCTAACCT

AAGAAG

(28 bp)

32

1

tel cen

Table. Characteristics of the nonrecurrent rearrangements in 17p

39

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

14The screen versions of these slides have full details of copyright and acknowledgements

Table. Reported complex gene rearrangements consistent with multiple FoSTeS events

40

Resolutions of complex genomic rearrangements

41

DNA Sequences reveal the complexity of ‘simple’ rearrangements

42

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

15The screen versions of these slides have full details of copyright and acknowledgements

43

Structural variation and disease

Clinical array rationale and design

• Targeted genomic regions

• Coalescence to whole genome coverage

Genomic rearrangement mechanisms

44

• Recombination: NAHR, NHEJ

• Replication: FoSTeS

De novo rearrangements and birth defects in neonates

Aneuploidy, Mendelian disease, complex traits: a continuum

Genomic rearrangements and sporadic disease

100 bp 104 105 106 107 108 109 bp101 102 103

DNA sequencing Chromosome bandingPFGE/FISH

45µ = (1/2)(1-w)B w = fitness; B = birth prevalence

Locus specific mutation rates (µ) 1.8 - 2.5 x 10-8

SNP

Array CGH

CNV1.7x10-6 – 1.2 x 10-4

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

16The screen versions of these slides have full details of copyright and acknowledgements

De novo locus specific mutationrates (µ) for genomic rearrangements

46Lupski (2007) Nature Genetics 39: S43-S47

Clinic indications at referral:

Patients and sampling:• 648 neonatal patients• Aged 28 days or younger• Blood samples collected March 2006 ~ September 2007

Xinyan Lu

Objective: to investigate genomic imbalance as a potential etiology in neonates

with birth defects

47

• Dysmorphic Features (DF)• Multiple Congenital Anomalies (MCA)• Congenital Heart Disease (CHD)• IUGR• Cleft Lip/Palate etc.• Suspected chromosomal syndrome;

Karyotype analysis identifies 21.6% with abnormalities

Xinyan Lu

Shah et al., (1990) Indian J Pediatr 57: 235-243Kenue et al., (1995) J Trop Pediatr 41: 77-80Goud et al., (2005) Saudi Med J 26: 1951-1957

Clinical indications and CMA clinically significant CNVs

48

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

17The screen versions of these slides have full details of copyright and acknowledgements

Structural variation and disease

Clinical array rationale and design

• Targeted genomic regions

• Coalescence to whole genome coverage

G i t h i

49

Genomic rearrangement mechanisms

• Recombination: NAHR, NHEJ

• Replication: FoSTeS

De novo rearrangements and birth defects in neonates

Aneuploidy, Mendelian disease, complex traits: a continuum

Variation in Daturadue to changes in chromosome number

50Station for experimental evolutionCold Spring Harbor L.I., N.Y.

The American NaturalistVol. 56: 16-31, 1922Dr. Albert Francis Blakeslee

Calvin B. Bridges (1936) the Bar “gene” a duplication

51Science 83: 210-211

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

18The screen versions of these slides have full details of copyright and acknowledgements

52

53

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“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

19The screen versions of these slides have full details of copyright and acknowledgements

Bridging the gap between chromosomal syndromes & Mendelian disorders

55

Duplication of APP

b) Copy number variationa) Trisomy 21

Promoters

c) SNPs in promoter regions

A. Alzheimer disease

Molecular mechanisms for chromosomal syndromes, Mendelian disease

and complex traits

56

APP SNPsAPP coding exons

B. Parkinson disease

SNCA

Duplication of SNCA

Triplication of SNCA

a) Copy number variation b) SNPs in promoter regions

SNCA coding exonsSNPs

Promoters

Genomic rearrangements and phenotypic traits

CNV CNV and disease Common traits Sporadic

Trai

t

57

susceptibilityp

& inherited disease

Examples: • HIV/CCL3L1• Lupus nephritis/FCGR3B

• Crohn / HBD-2

• Color blindness• Infertility• Hypertension• Autism• Schizophrenia• Olfactory variation?

• MR• (SMS, WBS, PWS/AS, DGS, Sotos)

• CMT• Hemophilia A• IP

Lupski and Stankiewicz (2005) PLOS Genet. 1(6):e49

Benign polymorphism

“Genomic Disorders: Mechanisms for Copy-Number Variation (CNV) and Clinical Implementation

of High-Resolution Genome Analysis”James R. Lupski, M.D., Ph.D.

20The screen versions of these slides have full details of copyright and acknowledgements

Conclusions• Both recombination (NAHR, NHEJ)

and replication (FoSTeS) mechanisms can generate CNV

• Elucidation of NAHR enables genome wide predictions of instability regions and reciprocal duplication/deletion events

58

• DNA replication FoSTeS mechanism may account for > 50% of duplication CNV at a given locus

• De novo rearrangements account for a significant fraction of sporadic traits including birth defects

The Lupski Lab et al.

59http://imgen.bcm.tmc.edu/molgen/lupski/

60