Cytogenetic Cytogenetic Rearrangements Mediated Rearrangements Mediated by Genomic Architecture by Genomic Architecture

in Meiosisin Meiosis

Cytogenetic Cytogenetic Rearrangements Mediated Rearrangements Mediated by Genomic Architecture by Genomic Architecture

in Meiosisin Meiosis

22q1122q11

A Closer Look at A Closer Look at 22q11.222q11.2

A Closer Look at A Closer Look at 22q11.222q11.2

Most frequent Most frequent microdeletion syndromemicrodeletion syndrome

Most common recurrent Most common recurrent balanced translocationbalanced translocation

Bisatellited marker Bisatellited marker chromosome of CESchromosome of CES

Numerous other Numerous other translocations at 22q11translocations at 22q11

The 22q11.2 Deletion The 22q11.2 Deletion Syndrome Syndrome (VCFS/DGS)(VCFS/DGS)

The 22q11.2 Deletion The 22q11.2 Deletion Syndrome Syndrome (VCFS/DGS)(VCFS/DGS)

Estimated frequency 1/3000-4000 Estimated frequency 1/3000-4000 live birthslive births

Autosomal Dominant Autosomal Dominant DisorderDisorder

Cleft or palatal anomalies Cleft or palatal anomalies ((69%69%)) Congenital heart defect Congenital heart defect ((74%74%)) Learning disabilityLearning disability Speech and language abnormalitiesSpeech and language abnormalities Psychiatric abnormalitiesPsychiatric abnormalities Immune dysfunctionImmune dysfunction Characteristic facial featuresCharacteristic facial features

DGCRDGCR22q11.222q11.2

ConstitutionalConstitutional t(11;22)t(11;22) ConstitutionalConstitutional t(11;22)t(11;22) Only known, recurrent, non-Only known, recurrent, non-Robertsonian translocation in manRobertsonian translocation in man

Seen in several hundred familiesSeen in several hundred families

Balanced carriers are Balanced carriers are phenotypically normalphenotypically normal

Risk of Supernumerary Risk of Supernumerary der(22)t(11;22) due to der(22)t(11;22) due to chromosomal malsegregationchromosomal malsegregation

47, XX or XY, +der(22) t(11;22)47, XX or XY, +der(22) t(11;22) Multiple malformation syndrome Multiple malformation syndrome Emanuel syndromeEmanuel syndrome

D22S427

D22S36

D22S941

D22S788

HCF2

CHKAD26

D22S801

Cen Tel

D22S75

BAC/PAC/Cosmid ContigBAC/PAC/Cosmid Contig

c106e4 c103a2 N25 c102g9 c68a1 c87f9 c2c9 c45c9c87h3

BAC 444p24 BAC 562f10PAC 194m11

BAC 32i11PAC 408l11

BAC 135h6

BAC 379n11PAC 413m7BAC 445f23

PAC 134n5

BAC 677f7BAC 293j14

BAC 291k7BAC 48m11

PAC 423n14

1.2 Mb DGCR cosmid contig

PAC 393h21

*

cH K89

low copy repeats = LCRslow copy repeats = LCRs

ZNF74

AA DDBB CC

22q11 Deletion and 22q11 Deletion and Rearrangement EndpointsRearrangement Endpoints22q11 Deletion and 22q11 Deletion and

Rearrangement EndpointsRearrangement Endpoints

t(11;22)t(11;22)CESCES CESCES

N25/HIRAN25/HIRA

D22S427 D22S36 D22S75 UFD1L D22S788 ZNF74 HCF2 CHKAD26 D22S936 Ki1165 D22S801 c61e11 c102d1CDC45L

TBX1

IGLL IGLLA A' B C D E F

DA

BA

A C

A' D

22/300 (7.3%)

5/300 (1.7%)5/300 (1.7%)

259/300 (86.3%)

Yamagishi et al.

CH01-242 CH98-205

Unbalanced translocations McQuade et al. Kurahashi et al.

CH04-18

CH98-18

Rauch et al.

CH97-221

CH03-029

CH03-064

CH03-162

CH02-008

Patient "g"

O'Donnell et al.

9/300 (3%)

Low Copy Repeats (LCRs) Low Copy Repeats (LCRs) in 22q11in 22q11

Structure and CompositionStructure and Composition

Low Copy Repeats (LCRs) Low Copy Repeats (LCRs) in 22q11in 22q11

Structure and CompositionStructure and Composition Modular StructureModular Structure

Modules range in size from 10-65 kbModules range in size from 10-65 kb

Modules contain previously identified Modules contain previously identified markers, truncated genes and markers, truncated genes and pseudogenespseudogenes

Variation in the content, orientation Variation in the content, orientation and organization of modules between and organization of modules between the different copiesthe different copies

AA

DD

CC

Genomic Organization of the Genomic Organization of the LCRs in 22q11.2LCRs in 22q11.2Genomic Organization of the Genomic Organization of the LCRs in 22q11.2LCRs in 22q11.2

AA

DD

CC

BBBB

LCRs and DeletionLCRs and Deletion Mechanism MechanismLCRs and DeletionLCRs and Deletion Mechanism Mechanism Normal Recombination EventNormal Recombination Event

A D

A

B

B C D

C

A

CA

B

B

C D

D

Crossover at LCR-DCrossover at LCR-D

Hypothetical Duplication or Hypothetical Duplication or Deletion MechanismDeletion Mechanism

Hypothetical Duplication or Hypothetical Duplication or Deletion MechanismDeletion Mechanism

““Misalignment followed by Recombination”Misalignment followed by Recombination”

A B C D

A DB C

A case of a deletion or A case of a deletion or duplication syndromeduplication syndrome

A D

Crossover between A & DCrossover between A & D

A B B C DADC

Detection of Rearranged Fragments

Detection of Rearranged Fragments

P1 P3 C 1 C2 C3 C4P2

A-B DeletionA-B DeletionProbed with ZNF74Probed with ZNF74

650 kb

145 kb

..

NotI

NotI

NotI

D22S427

ZNF74

D22S788

NotI

650 kb

NotI

D22S427

ZNF74

A B

145 kb

PCR Analysis of LCRA-LCRB Junction Fragment

PCR Analysis of LCRA-LCRB Junction Fragment

1 2 3 6 1 2 3 6 1 2 344 55 44 55 44 55 6M M

NormalNormal

DeletedDeleted

BB

Not I SiteNot I Site

PCR MarkerPCR Marker

145 kb

Lane 4Lane 4

650 kb

Lane 5Lane 5

ZNF74

ZNF74

444p24Sp6444p24Sp637g5T3

37g5T3

A B

ZNF74 444p24Sp6 37g5T3

Parent of Origin Parent of Origin DeterminationsDeterminationsParent of Origin Parent of Origin DeterminationsDeterminations

Maternal DeletionsMaternal Deletions36 36

Paternal DeletionsPaternal Deletions 3333

Number of patients Number of patients examined = 69examined = 69

Examining the Examining the Mechanism of DeletionMechanism of Deletion

Examining the Examining the Mechanism of DeletionMechanism of Deletion

Haplotype reconstruction Haplotype reconstruction

3 generation families3 generation families

Markers flanking the Markers flanking the deletion regiondeletion region

Genescan analysis Genescan analysis

Results of Studies Results of Studies to Dateto DateResults of Studies Results of Studies to Dateto Date

20 informative families20 informative families

19 consistent with an interchromosomal 19 consistent with an interchromosomal crossover event - much greater than expected crossover event - much greater than expected for meiotic distancefor meiotic distance

1 consistent with an intrachromosomal event1 consistent with an intrachromosomal event normal 22 only has 2 interchromosomal events normal 22 only has 2 interchromosomal events in 15 examined - more consistent with in 15 examined - more consistent with meiotic mapping studiesmeiotic mapping studies

no evidence for phenotypic differences no evidence for phenotypic differences related to heart or palate based on parental related to heart or palate based on parental origin of deletion in any of the familiesorigin of deletion in any of the families

FISH to Detect an FISH to Detect an InversionInversionFISH to Detect an FISH to Detect an InversionInversion

3 Mb Deletion Region in 22q11.2

A DD22S427 CHKAD26

c106e4 87h3 CHKAD26

N25 D22S788 ZNF74

B

Summary of 22q11.2 Summary of 22q11.2 DeletionsDeletionsSummary of 22q11.2 Summary of 22q11.2 DeletionsDeletions

No major parent of origin effects - deletion No major parent of origin effects - deletion or phenotypeor phenotype

Majority are Majority are de novode novo 3 Mb deletions 3 Mb deletions

No genotype-phenotype correlations- No genotype-phenotype correlations- phenotype is quite variablephenotype is quite variable

Unusual number of interchromosomal exchangesUnusual number of interchromosomal exchanges unequal crossing over involving segmental unequal crossing over involving segmental duplicationsduplications

other meiotic studies indicate proximal crossovers other meiotic studies indicate proximal crossovers are infrequentare infrequent

Inversions do not predispose to the deletion Inversions do not predispose to the deletion or the phenotype in absence of a deletionor the phenotype in absence of a deletion

Isolation and analysis of Isolation and analysis of the the

Recurrent t(11;22)Recurrent t(11;22)(q23;q11), t(17;22) and (q23;q11), t(17;22) and

t(A;22)t(A;22)

Isolation and analysis of Isolation and analysis of the the

Recurrent t(11;22)Recurrent t(11;22)(q23;q11), t(17;22) and (q23;q11), t(17;22) and

t(A;22)t(A;22)

LCR-B on 22q and the LCR-B on 22q and the Constitutional t(11;22)Constitutional t(11;22)LCR-B on 22q and the LCR-B on 22q and the

Constitutional t(11;22)Constitutional t(11;22)

N N

AA BB CC DDcentromere

telomere

(AT)n (AT)n(AT)n ZNF74v

t(11;22)t(11;22)

b562f10b444p24

cHK89

duplication modules

90 kb gap90 kb gap

N41

PATTR at 11q23 and Stem PATTR at 11q23 and Stem Loop Cruciform StructuresLoop Cruciform StructuresPATTR at 11q23 and Stem PATTR at 11q23 and Stem Loop Cruciform StructuresLoop Cruciform Structures

Inverted Inverted Repeats Repeats

(palindrome(palindrome))

TA

TC

TG

A

TA

G

A

CA T

A

TCTGTATCTGT

A

CA

G

ATA

CA

G

A

T

TA

CA

G

A

A

TA

G

A

C

TA

TC

TG

AGA

CA

TA

GA

C

ATG

TC

T

A

TGTCTATGTC

T

TA

ATT C T G

A G A C A T A G A C

T A T C T G T A T C T GA

T A G A C AT TGTCT

ACAGATACAGA

ATGTCTATGTCT

TACAGA

A

TA

G

A

C

T

A

TC

TG

A

TC

TG

TA

G

A

CA

TG

T

C

T

TA

C

A

GA

TA

C

A

G

A

A

TGT

C

TA

TG

T

C

T

TA

C

A

GA

A

TA

G

A

C

TA

T

C

TG

ATT

G

A

T

C

TG

TA

TC

T

A

CA

T

T

C

T

A

T

C

T

A T

AG

A

CA

TAG

A

C

T

G

A

TA

G

ACA

TG

T

C

T

T

A

A

A

G

A

A

TG

T

C

TA

TG

T

C

T

A

A

AA

TA

A

T

T

T

T

T

T

A

A

A

G

G

G

T

T

C

Genomic Instability Mediates Genomic Instability Mediates the t(11;22)the t(11;22)Genomic Instability Mediates Genomic Instability Mediates the t(11;22)the t(11;22)

Chromosome 22Unclonable

BacteriaTranslocation

Human Genome

Human Genome

Deletion

Bacteria

Chromosome 11

Analysis of the t(11;22) Analysis of the t(11;22) Breakpoint (BP)Breakpoint (BP)Analysis of the t(11;22) Analysis of the t(11;22) Breakpoint (BP)Breakpoint (BP) > 42 independent BPs have been examined - PATRRs > 42 independent BPs have been examined - PATRRs at 11q23 and 22q11 BPs at 11q23 and 22q11 BPs

Breakpoints in all families are almost identical Breakpoints in all families are almost identical with very limited variationwith very limited variation

Families are of various ethnic and racial Families are of various ethnic and racial backgrounds backgrounds i.e., implicates a similar mechanism rather than a i.e., implicates a similar mechanism rather than a founder effectfounder effect

BPs occur at the tip of the palindrome with BPs occur at the tip of the palindrome with small, symmetrical deletionssmall, symmetrical deletions

suggests that there is likely a palindrome mediated suggests that there is likely a palindrome mediated mechanism responsible for the recurrent nature of the mechanism responsible for the recurrent nature of the translocationtranslocation

Analysis of Analysis of t(17;22)s in the t(17;22)s in the

NF1 GeneNF1 Gene

Analysis of Analysis of t(17;22)s in the t(17;22)s in the

NF1 GeneNF1 Gene

exon 31exon 31 exon 32exon 32centromerecentromere telomeretelomere

********************************************************************************

proximal proximal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATA--------TATATATATATA 52CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATA--------TATATATATATA 52distal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATACATATATATATATATATATA 60distal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATACATATATATATATATATATA 60 **************************************** **************************************************** ************

proximalproximal TATGTATATAATTATATAGGATTATATGTAGGATTATATTTATATGTATATAATTATATAGGATTATATGTAGGATTATATTTA 9494distaldistal TATGTATATAATTATATAGGATTATATGTAGGATTATATTA- 101TATGTATATAATTATATAGGATTATATGTAGGATTATATTA- 101

209 bp209 bp

PATRRPATRR

195 195 bpbp

Unexpectedly High Rate of Unexpectedly High Rate of t(11;22)s in Sperm from t(11;22)s in Sperm from

Normal MalesNormal Males

Unexpectedly High Rate of Unexpectedly High Rate of t(11;22)s in Sperm from t(11;22)s in Sperm from

Normal MalesNormal Males PCR performed on sperm samples from four normal PCR performed on sperm samples from four normal males was positive for the t(11;22) PCR product males was positive for the t(11;22) PCR product

The estimated frequencies are : The estimated frequencies are : 1.24 - 9.46x101.24 - 9.46x10-5-5

Testicular DNA: Testicular DNA: positivepositive

Twenty normal lymphoblast or fibroblast DNAs: Twenty normal lymphoblast or fibroblast DNAs: negativenegative

Lymphoblasts from patients with Bloom syndrome Lymphoblasts from patients with Bloom syndrome or ataxia-telangiectasia:or ataxia-telangiectasia: negativenegative

PCR for t(17;22) in same sperm samples: PCR for t(17;22) in same sperm samples: negativenegative

Polymorphism of the PATRR affects translocation Polymorphism of the PATRR affects translocation frequencyfrequency

Polymorphisms of the 11q Polymorphisms of the 11q PATRRPATRRPolymorphisms of the 11q Polymorphisms of the 11q PATRRPATRR

Allele typeAllele type NumberNumber Frequency(%)Frequency(%) StructureStructure

Long (L)Long (L) 343343 87.187.1

Short (S)Short (S) S1S1 2020 (40.0)(40.0)

55 (10.0)(10.0)

S3S3 2424 (48.0)(48.0)

S4S4 11 (2.0)(2.0)

5050 12.712.7

11 0.30.3

TotalTotal 394394 100100

Extra-long (EL)Extra-long (EL)

SubtotalSubtotal

S2S2

Deletion/insertion type polymorphisms have been identifiedDeletion/insertion type polymorphisms have been identified

6.81 x 106.81 x 10-7-7

<1.69 x 10<1.69 x 10-7-7

1.07-2.27 x 101.07-2.27 x 10-5-5

<3.05 x 10<3.05 x 10-7-7

Translocation Translocation FrequencyFrequency

1.20-2.93 x 101.20-2.93 x 10-6-6

1.55-1.71 x 101.55-1.71 x 10-6-6

Kato et al., Science 311:971 2006Kato et al., Science 311:971 2006

AB

Does the 11q23 PATRR Adopt a Cruciform Does the 11q23 PATRR Adopt a Cruciform Configuration?Configuration?

Atomic Force MicroscopyAtomic Force Microscopy

Does the 11q23 PATRR Adopt a Cruciform Does the 11q23 PATRR Adopt a Cruciform Configuration?Configuration?

Atomic Force MicroscopyAtomic Force Microscopy

Other Translocations Other Translocations to 22q11.2to 22q11.2Other Translocations Other Translocations to 22q11.2to 22q11.2 Numerous translocations have been describedNumerous translocations have been described None of the others are recurrent None of the others are recurrent translocationstranslocations

Question: Are they located in LCR-B?Question: Are they located in LCR-B? Analysis to the sequence level to determineAnalysis to the sequence level to determine

Is a PATRR a consistent finding at the BP?Is a PATRR a consistent finding at the BP? Do the different partner chromosomes share Do the different partner chromosomes share sequence homologysequence homology with one anotherwith one another with 22q?with 22q?

Do they demonstrate any similarities to one Do they demonstrate any similarities to one another with respect to location or organization another with respect to location or organization of the BPsof the BPs

Stem Loop Structures of LCR-B Stem Loop Structures of LCR-B and its Partnersand its Partners

Stem Loop Structures of LCR-B Stem Loop Structures of LCR-B and its Partnersand its Partners

22q11.2 LCR-B22q11.2 LCR-B(594 bp)(594 bp)

100

500 400

200

BP Region

11q2311q23(445 bp)(445 bp)

BP Region100

400 300

17q1117q11(205 bp)(205 bp)

4080

120160200

4q35.14q35.1(1072 bp)(1072 bp)

100 2001000 900

300

600800

1000

600

100

500

300

900

0/1200 700

1100

200

400

800

Deduced 1p21.2 AT-rich Deduced 1p21.2 AT-rich BreakpointBreakpointSequence Sequence

[t(1;22) patient][t(1;22) patient]

600

100500300

900

800

0/1200

700

1000

1100

200

400

Reference 1p21.2Reference 1p21.2SequenceSequence

(RP4-600M23)(RP4-600M23)

Stem Loop Structure of Stem Loop Structure of t(1;22) BPt(1;22) BP

Stem Loop Structure of Stem Loop Structure of t(1;22) BPt(1;22) BP

Non-LCR-B Translocations Non-LCR-B Translocations No Palindromic Sequences - No No Palindromic Sequences - No

Stem LoopsStem Loops

Non-LCR-B Translocations Non-LCR-B Translocations No Palindromic Sequences - No No Palindromic Sequences - No

Stem LoopsStem Loopst(2;22))

2q142q14 22q11.222q11.2t(10;22)

10q2610q26 22q11.222q11.2 15q2615q26 22q12.122q12.1t(15;22)

t(21;22)21p1221p12 22q11.222q11.2 2q37.22q37.2 Xp21.1Xp21.1

t(X;2) t(X;4)4q35.24q35.2 Xp21.1Xp21.1

Analysis of Recombination Analysis of Recombination and Proximity in Meiosisand Proximity in MeiosisAnalysis of Recombination Analysis of Recombination and Proximity in Meiosisand Proximity in Meiosis

LEGENDBlue = CRESTRed = SCP3Green = MLH1

Fuschia = c87f9Gold - 442e11

Comparison Between Comparison Between Oocytes and Oocytes and

SpermatocytesSpermatocytes

Comparison Between Comparison Between Oocytes and Oocytes and

SpermatocytesSpermatocytes

00

200200

400400

600600

800800

10001000

12001200

14001400

16001600

18001800

11 33 55 77 99 1111 1313 1515 1717 1919 2121 2323 2525 2727 2929 3131 3333 3535 3737 3939 4141 4343 4545 4747 4949

DistanceDistance

Oocyte or Spermatocyte #Oocyte or Spermatocyte #

11q-22q

6q-22q

11q-22q

6q-22q

spermatocytesspermatocytes oocytesoocytes

Are the Breakpoints at Are the Breakpoints at Recombination “Hot-Recombination “Hot-

Spots?”Spots?”

Are the Breakpoints at Are the Breakpoints at Recombination “Hot-Recombination “Hot-

Spots?”Spots?”

SummarySummarySummarySummary

The “genomic instability” of 22q11.2 is The “genomic instability” of 22q11.2 is presumed to be based on the presence of presumed to be based on the presence of multiple LCRs or segmental duplicationsmultiple LCRs or segmental duplications

Recurrent 22q11.2 deletion endpoints coincide Recurrent 22q11.2 deletion endpoints coincide with the low copy repeats (LCRs) on chromosome with the low copy repeats (LCRs) on chromosome 22 22

Inversions are not a common feature that Inversions are not a common feature that predisposes to the deletionpredisposes to the deletion

Interchromosomal recombination occurs in most Interchromosomal recombination occurs in most de novode novo deletions - not the favored location deletions - not the favored location for recombination eventsfor recombination events

Summary Summary ContinuedContinuedSummary Summary ContinuedContinued LCR-B is the location of the t(11;22) breakpoint as LCR-B is the location of the t(11;22) breakpoint as

well as many other translocations involving 22q11, well as many other translocations involving 22q11, but not all of thembut not all of them

A palindrome mediated mechanism is responsible for A palindrome mediated mechanism is responsible for the translocations occurring in LCR-Bthe translocations occurring in LCR-B

Other 22q11 translocations appear to be more randomOther 22q11 translocations appear to be more random

LCR-B translocation BPs exhibit a propensity to LCR-B translocation BPs exhibit a propensity to form secondary structure apparently related to form secondary structure apparently related to their rate of recurrence and perhaps polymorphismstheir rate of recurrence and perhaps polymorphisms

Constitutional translocations seem to occur Constitutional translocations seem to occur between sequences of similar melting temperature between sequences of similar melting temperature and/or secondary structure.and/or secondary structure.

AcknowledgemeAcknowledgementsntsAcknowledgemeAcknowledgementsnts

Anthony Gotter Anthony Gotter

Sulagna SaittaSulagna Saitta

Tamim Shaikh Tamim Shaikh

Hiroki KurahashiHiroki Kurahashi

Stacy HarrisStacy Harris

April HackerApril Hacker

Jill Yersak Jill Yersak

Mandy BookMandy Book

Danielle ConfortoDanielle Conforto

Debbie DriscollDebbie Driscoll

Elaine ZackaiElaine Zackai

Donna McDonald-Donna McDonald-McGinnMcGinn

Mellissa TonnesonMellissa Tonneson

Livija CelleLivija Celle

Jason CatanzaroJason Catanzaro

Jeanne MansonJeanne Manson

Marcia BudarfMarcia Budarf

MaimonCohMaimonCohenen

Terry Terry AshleyAshley

Ann GaethAnn Gaeth

Harker Harker RhodesRhodes

Tatsushi Tatsushi TodaToda

Takema Takema KatoKato

LocaLocall

Outside Outside InstitutionsInstitutions