Chromosomal Rearrangements, Candidate Genes and

Congenital Heart Defects

Chromosomal Rearrangements, Candidate Genes and

Congenital Heart DefectsBeverly S. Emanuel, Ph.D.Beverly S. Emanuel, Ph.D.

Ken Ryan, Ph.D.Ken Ryan, Ph.D.

Sulagna Saitta, M.D., Ph.D. Sulagna Saitta, M.D., Ph.D.

Eric Weinberg, Ph.D.Eric Weinberg, Ph.D.

Anthony Gotter, Ph.D.Anthony Gotter, Ph.D. Julia BrownJulia Brown

Manjunath Nimmakayalu, Ph.D.Manjunath Nimmakayalu, Ph.D. April Schrank-HackerApril Schrank-Hacker

Reza Jalali, Ph.D.Reza Jalali, Ph.D. Christine SuppaChristine Suppa

Smita Purandare, M.D., Ph.D.Smita Purandare, M.D., Ph.D. Ron O’Connor Ron O’Connor

Brianne O’Loughlin Brianne O’Loughlin

Specific AimsSpecific Aims Identify and characterize CRs in patients with CHD by high-resolution cytogenetics and Identify and characterize CRs in patients with CHD by high-resolution cytogenetics and

molecular cytogenetic analysismolecular cytogenetic analysis

Identify and fine map the translocations or rearrangements using the existing genomic Identify and fine map the translocations or rearrangements using the existing genomic resourcesresources

Develop PCR-based mapping strategies using the human genomic sequence to identify Develop PCR-based mapping strategies using the human genomic sequence to identify the translocation BPsthe translocation BPs

Characterize the genomic DNA from normal chromosomes at the BPs in order to Characterize the genomic DNA from normal chromosomes at the BPs in order to identify mechanisms of rearrangementidentify mechanisms of rearrangement

Identify candidate genes disrupted or deleted at the translocation BPs as candidates for Identify candidate genes disrupted or deleted at the translocation BPs as candidates for early cardiac morphogenesisearly cardiac morphogenesis

Determine whether mutations in the candidate genes are associated with the cardiac Determine whether mutations in the candidate genes are associated with the cardiac defect in other patients with CHDdefect in other patients with CHD

HypothesesHypotheses Apparently balanced translocations will help pinpoint Apparently balanced translocations will help pinpoint

gene(s) that are important in early cardiac gene(s) that are important in early cardiac morphogenesismorphogenesis

Chromosomal additions or interstitial deletions will Chromosomal additions or interstitial deletions will assist in the identification of dosage sensitive genes assist in the identification of dosage sensitive genes important in cardiac developmentimportant in cardiac development

The expression or function of some genes is disrupted The expression or function of some genes is disrupted or modified by genomic interruption, chromosomal or modified by genomic interruption, chromosomal position or dosage imbalanceposition or dosage imbalance

Specific Aim 1Specific Aim 1 Identify and characterize CRs in patients with CHD by high-resolution Identify and characterize CRs in patients with CHD by high-resolution

cytogenetics and molecular cytogenetic analysiscytogenetics and molecular cytogenetic analysis over 411 SCCOR patients studied to date by HRB G-band analysisover 411 SCCOR patients studied to date by HRB G-band analysis

Includes some old (SCOR) and new (SCCOR) patients Includes some old (SCOR) and new (SCCOR) patients

22q11.2 deletions excluded from karyotype study22q11.2 deletions excluded from karyotype study

A total of 5 deleteds in 2005A total of 5 deleteds in 2005

Still only 3 cytogenetically abnormal karyotypesStill only 3 cytogenetically abnormal karyotypes

t(13;16) (t(13;16) (de novode novo))

t(1;9) (t(1;9) (de novode novo))

46,XX, 13 p46,XX, 13 p+ + - ruled out as a variant- ruled out as a variant

50 patients examined for telomere anomalies50 patients examined for telomere anomalies

No telomere anomalies detectedNo telomere anomalies detected

Cell Line46,XY,t(13;16)(q14.2;q21)

(TGA CH01-069/798-ACH01-069/798-A)

Cell Line46,XY,t(13;16)(q14.2;q21)

(TGA CH01-069/798-ACH01-069/798-A)

April Schrank-HackerApril Schrank-Hacker

13q14 (RP11-54G17; 110,249 bp)13q14 (RP11-54G17; 110,249 bp)

SETDB2SETDB2

RP11-54G17 FISH ProbeRP11-54G17 FISH Probe

MO25-LIKESETDB2

13q14 : MO2L (MO25-Like)13q14 : MO2L (MO25-Like)

Function unknownFunction unknown

Contains ARM repeats and a zinc fingerContains ARM repeats and a zinc finger

MO25 = a scaffold protein for tumor suppressor protein MO25 = a scaffold protein for tumor suppressor protein kinase, LKB1 and STRADkinase, LKB1 and STRAD4K (forming a regulatory 4K (forming a regulatory complex; Milburn 2004)complex; Milburn 2004)

ARM = ß-catenin => Wnt signalingARM = ß-catenin => Wnt signaling

ß-catenin associates with ion channels (Lesage 2004) in ß-catenin associates with ion channels (Lesage 2004) in 9th ARM repeat (Lin2/CASK)9th ARM repeat (Lin2/CASK)

Fine Mapping the t(13;16) Breakpoint Fine Mapping the t(13;16) Breakpoint for Junction Fragment Sequencingfor Junction Fragment Sequencing

Fine Mapping the t(13;16) Breakpoint Fine Mapping the t(13;16) Breakpoint for Junction Fragment Sequencingfor Junction Fragment Sequencing

FISH using strategically designed PCR-FISH using strategically designed PCR-generated probesgenerated probes

Traditional Southern mappingTraditional Southern mapping

Adapter-ligated PCRAdapter-ligated PCR

Chromosome CaptureChromosome Capture

SETDB2CC DD EEBBAA

Mo25-Like

RP11-54G17 FISH Probe

13q14.2 Breakpoint is in the Coding Region of 13q14.2 Breakpoint is in the Coding Region of the MO25-Like Genethe MO25-Like Gene

der (13) 13

der (16)

13

Tony Gotter and Manju NimmakayaluTony Gotter and Manju Nimmakayalu

Probes Used For Southern Blot Determination Probes Used For Southern Blot Determination of the 13q14.2 Breakpointof the 13q14.2 Breakpoint

Probes Used For Southern Blot Determination Probes Used For Southern Blot Determination of the 13q14.2 Breakpointof the 13q14.2 Breakpoint

FISH probes:FISH probes:

Mo25-Like7 6 5891011 4

Southern probesSouthern probes:Nco I

NheI

Spe I

Xba I

BamHI

Bstx I

EcoR I

HIND III

BamI

NcoI

NheI

SpeI

XbaI

BstxI

EcoRI

HINDIII

NcoI

SpeI

NheI

XbaI

BamHI

BstxI

EcoRI

HINDIII

NcoI

NheI

SpeI

XbaI

BamHI

BstxI

EcoRI

HINDIII

ATGATGATGATG

AA CC

Tony Gotter and Julia BrownTony Gotter and Julia Brown

CCAA

Mo25-LikeMo25-Like

RP11-54G17 FISH Probe

7

FISH probesFISH probes

6 5

ATGATG

891011

E7E7Southern Probe:Southern Probe:

Southern Fragments:Southern Fragments:(breakpoint excluded from this (breakpoint excluded from this ~20kb region)~20kb region)

~17kb~17kb~27kb~27kb

Possible regions left examine Possible regions left examine for the BP:for the BP:

7 6 5891011

Southern Analysis with Exon 7 of MO25LSouthern Analysis with Exon 7 of MO25L

C1C1 C1C1 C1C1 C1C1C2C2 C2C2 C2C2 C2C2PtPt PtPt PtPt PtPt

NcoINcoI NheINheI SpeISpeI XbaIXbaI

C1C1 C1C1 C1C1 C1C1C2C2 C2C2 C2C2 C2C2PtPt PtPt PtPt PtPt

BamHIBamHI BstxIBstxI EcoRIEcoRI HINDIIIHINDIII

23.123.1

9.49.4

6.66.6

23.123.1

9.49.4

6.66.6

4.04.0

3.03.0

Genomic Southerns Probed Genomic Southerns Probed for Exon 10 Restriction Fragmentsfor Exon 10 Restriction Fragments

Tony Gotter and Julia BrownTony Gotter and Julia Brown

Genomic Southerns Probed Genomic Southerns Probed for Exon 11 Restriction Fragmentsfor Exon 11 Restriction Fragments

C1C1 C1C1 C1C1 C1C1C2C2 C2C2 C2C2 C2C2PtPt PtPt PtPt PtPt

BamHIBamHI BstxIBstxI EcoRIEcoRI HINDIIIHINDIII

C1C1 C1C1 C1C1 C1C1C2C2 C2C2 C2C2 C2C2PtPt PtPt PtPt PtPt

NcoINcoI NheINheI SpeISpeI XbaIXbaI

23.123.1

9.49.4

6.66.6

23.123.1

9.49.4

6.66.6

Tony Gotter and Julia BrownTony Gotter and Julia Brown

Fine Mapping and Cloning the t(13;16) Fine Mapping and Cloning the t(13;16) Junctions By Adapter-Ligated PCRJunctions By Adapter-Ligated PCR

(GeneWalker System, BD Biosciences)(GeneWalker System, BD Biosciences)

Fine Mapping and Cloning the t(13;16) Fine Mapping and Cloning the t(13;16) Junctions By Adapter-Ligated PCRJunctions By Adapter-Ligated PCR

(GeneWalker System, BD Biosciences)(GeneWalker System, BD Biosciences)

Digest with blunt R.E. Adapter Ligation

Blunt-End Restriction DigestBlunt-End Restriction Digest(EcoRV)(EcoRV)

+ + + + Adapter LigationAdapter Ligation

Sequence Abnormally SizedSequence Abnormally SizedFragmentsFragments

t(13;16) Junction Sequencet(13;16) Junction Sequence

PCRPCR

Normal 13Normal 13 der (13)der (13)

EcoRVEcoRV EcoRVEcoRV

16q16q 13q13q

EcoRVEcoRVEcoRVEcoRV

Tony Gotter and Carolina MartinezTony Gotter and Carolina Martinez

Mutation Analysis MO25LMutation Analysis MO25L

10 patients with dTGA10 patients with dTGA PCR for coding exonsPCR for coding exons Several polymorphisms in exon 5Several polymorphisms in exon 5 No mutations found No mutations found Additional patients to be sequencedAdditional patients to be sequenced

Looking For MO25-Like Mutations in Patients With d-TGA

Looking For MO25-Like Mutations in Patients With d-TGA

10 patients were chosen based on cardiac phenotype 10 patients were chosen based on cardiac phenotype t(13;16) patient (d-TGA)t(13;16) patient (d-TGA)

All coding exons (4 to 11) PCR amplified using primers flanking the All coding exons (4 to 11) PCR amplified using primers flanking the exons exons

Sequences were compared to known database sequence, CEPH DNA Sequences were compared to known database sequence, CEPH DNA and the t(13;16) patientand the t(13;16) patient

Differences were seen in CEPH and patient vs. database sequence:Differences were seen in CEPH and patient vs. database sequence:

13q14.213q14.2

ATGATG

77 66 55889910101111 44

MO25-Like Exon 5 Polymorphisms in MO25-Like Exon 5 Polymorphisms in Patients With d-TGAPatients With d-TGA

MO25-Like Exon 5 Polymorphisms in MO25-Like Exon 5 Polymorphisms in Patients With d-TGAPatients With d-TGA

Exon 5Exon 5

(A/G 509*)(A/G 509*)

Exon 5Exon 5

(A/G 560*)(A/G 560*)

16 bp Insert16 bp Insert

(Intron 5 [+ 60])(Intron 5 [+ 60])

Database: Database: AA AA --

CEPH (CD-9):CEPH (CD-9): A/G (Het)A/G (Het) A/G (Het)A/G (Het) +/- (Het)+/- (Het)

Patient Samples:Patient Samples: A/A (Hom) 2/10A/A (Hom) 2/10 A/A (Hom) 2/10A/A (Hom) 2/10 -/- (Hom) 2/10-/- (Hom) 2/10

G/G (Hom) 4/10G/G (Hom) 4/10 G/G (Hom) 4/10G/G (Hom) 4/10 +/+ (Hom) 1/10+/+ (Hom) 1/10

A/G (Het) 4/10A/G (Het) 4/10 A/G (Het) 4/10A/G (Het) 4/10 +/- (Het) 7/10+/- (Het) 7/10

Human HEK293 Cells Manipulated to Express Human HEK293 Cells Manipulated to Express MO25-Like in a Tetracycline-Inducible MannerMO25-Like in a Tetracycline-Inducible MannerHuman HEK293 Cells Manipulated to Express Human HEK293 Cells Manipulated to Express MO25-Like in a Tetracycline-Inducible MannerMO25-Like in a Tetracycline-Inducible Manner

Tetracycline-inducible Mo25-Like-expressing cells can be used in gene Tetracycline-inducible Mo25-Like-expressing cells can be used in gene expression analysis (microarray) to identify MO25-Like target genesexpression analysis (microarray) to identify MO25-Like target genes

Anti-MO25L antibody can be used to study the tissue and cellular Anti-MO25L antibody can be used to study the tissue and cellular distribution of the endogenous proteindistribution of the endogenous protein

0 16 24 0 16 24

Anti-myc Anti-Mo25Like

Tetracycline (hrs):

hMO25L-myc

Anti-Mo25Like Antisera Recognizes Tetracycline-Induced Protein (right panel)Anti-Mo25Like Antisera Recognizes Tetracycline-Induced Protein (right panel)

Tony GotterTony Gotter

Ongoing Investigation of Mo2L in Model Organisms

Ongoing Investigation of Mo2L in Model Organisms

xMo2L and zMo2L cDNA clones obtained xMo2L and zMo2L cDNA clones obtained and in sequencing; xMo2L verifiedand in sequencing; xMo2L verified

ISH reagents purchased, stocks preparedISH reagents purchased, stocks prepared

xMo2L is Expressed in Xenopus Development

xMo2L is Expressed in Xenopus Development

Carrie DasconioCarrie Dasconio

Ongoing ExperimentsOngoing Experiments

Over-expression of xMo2L (400 pg mRNA Over-expression of xMo2L (400 pg mRNA injected) in the ventral presumptive mesoderm injected) in the ventral presumptive mesoderm may inhibit gastrulation (S4-87)may inhibit gastrulation (S4-87)

Over-expression of xMo2L in the animal pole has Over-expression of xMo2L in the animal pole has no effectno effect

xMo2L does not activate the early Wnt pathway xMo2L does not activate the early Wnt pathway in Xenopusin Xenopus

Future ExperimentsFuture Experiments

Higher dose of xMo2LHigher dose of xMo2L Animal pole explant assaysAnimal pole explant assays

Regrown Cell Line46,XY,t(1;9)(q23.3;q32)

(Tetralogy of Fallot)

Regrown Cell Line46,XY,t(1;9)(q23.3;q32)

(Tetralogy of Fallot)

April Schrank-HackerApril Schrank-Hacker

Candidate Gene Encoded at the 1q23.3 BPCandidate Gene Encoded at the 1q23.3 BP

RP11-2a6 FISH ProbeRP11-2a6 FISH Probe

LIMX1ALIMX1A

RP11-30i17 (1q21.1)

RP11-79m15 (1q23.3)

RP11-90a11 (1q23.3)

RP11-449b2s3 (1q23.3)

RP11-77g8 (1q23.3)

RP11-503n16 (1q23.3)

RP11-403p14 (1q23.3)

RP11-2a6

RP11-38c18 (1q23.3)

RP11-80l8 (1q23.3)

RP11-449d21 (1q24.1)

RP11-177m16 (1q25)

RP11-90c19 (1q25.2)

RP11-89j22 (1q25.3)

RP11-91e21 (1q25.3)

split signal

Candidate Genes Encoded at the 9q31.3 Breakpoint

RP11-202g18 (9q32) -----Split signal (BP)RP11-202g18 (9q32) -----Split signal (BP)

Candidate Genes Encoded at the 9q31.3 Breakpoint

RP11-202g18 (9q32) -----Split signal (BP)RP11-202g18 (9q32) -----Split signal (BP)

RP11-202g18 FISH ProbeRP11-202g18 FISH Probe

(OR2K2)(OR2K2)EDG2EDG2A B C D

Cloned PCR Generated FISH ProbesCloned PCR Generated FISH ProbesBP to be better definedBP to be better defined

Tony Gotter and Manju NimmakayaluTony Gotter and Manju Nimmakayalu

FISH to Define 9q BPFISH to Define 9q BP

278d5(g)/140a9 (g) (maps to 1p36)278d5(g)/140a9 (g) (maps to 1p36)

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

202g18202g18278d5278d5

der(1)der(1)

der(9)der(9)

Spanning cloneSpanning clone

9q139q13

Manju NimmakayaluManju Nimmakayalu

99

11

Human LPA1 (EDG2)Human LPA1 (EDG2)

G -protein receptor for lysophosphotidic acidG -protein receptor for lysophosphotidic acid

Cell signaling and migration in neurogenesisCell signaling and migration in neurogenesis

Conserved in human, mouse, xenopus, zebrafish Conserved in human, mouse, xenopus, zebrafish

cDNA isolated from human lung library, full length is 1.5 cDNA isolated from human lung library, full length is 1.5 kb; alternatively spliced forms presentkb; alternatively spliced forms present

Northern analysis shows wide expression, with highest Northern analysis shows wide expression, with highest expression in brain followed by heart, not expressed in PBLexpression in brain followed by heart, not expressed in PBL

rtPCR of Human Fetal PanelrtPCR of Human Fetal Panel

brai

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lung live

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kidn

ey

hear

t

sple

en

thym

us

Ske

leta

l mus

cle

Sulagna Saitta, Brianne O’LoughlinSulagna Saitta, Brianne O’Loughlin

rtPCR of Human Cardiovascular PanelrtPCR of Human Cardiovascular Panel

Rig

ht A

triu

m

Adu

lt h

eart

Lef

t Ven

tric

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Rig

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entr

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Lef

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apex

aort

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Fet

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eart

Dex

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auri

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Sin

istr

a au

ricl

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Inte

rvet

ricu

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sept

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Atr

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Sulagna Saitta, Brianne O’LoughlinSulagna Saitta, Brianne O’Loughlin

EDG2/LPA1 Experimental Progress/PlanEDG2/LPA1 Experimental Progress/Plan

Sulagna Saitta, Ron O’Connor, Brianne O’LoughlinSulagna Saitta, Ron O’Connor, Brianne O’Loughlin

Primers for PCR developed in the intron-exon junctions (not Primers for PCR developed in the intron-exon junctions (not including potential splicing sequences) including potential splicing sequences)

Multiple patients have been amplified for sequencing Multiple patients have been amplified for sequencing (translocation proband had TOF) (translocation proband had TOF)

Patients Screened for EDG2 Mutations (Exons 2-5)

Patients Screened for EDG2 Mutations (Exons 2-5)

Patient diagnosisPatient diagnosis Number of patients Number of patients screenedscreened

Tetralogy of FallotTetralogy of Fallot 1212

Non-deleted VCFSNon-deleted VCFS 45-5045-50

Opitz syndromeOpitz syndrome 55

Note: 8 of the ND-VCFS patients have TOFNote: 8 of the ND-VCFS patients have TOF

Sulagna Saitta, Brianne O’LoughlinSulagna Saitta, Brianne O’Loughlin

Single Base Pair Changes FoundSingle Base Pair Changes Found (in ND-VCFS Patients) (in ND-VCFS Patients)

Single Base Pair Changes FoundSingle Base Pair Changes Found (in ND-VCFS Patients) (in ND-VCFS Patients)

Patient # Dx Exon Nucleotide Codon Amino AcidCH97-195 TOF 4A 520 G -> T UGG-> UGU Trp -> CysCH97-042 NDVCFS 4B 655 T -> C AAU -> AAC Asn -> AsnCH97-179 NDVCFS 4B 782 G -> A GAG -> GAA Glu -> GluCH00-090 NDVCFS 5 941 C -> T CGC -> CGU Arg -> Arg

Identical single base pair change found in unaffected relative of CH97-179Identical single base pair change found in unaffected relative of CH97-179 CH00-090 change found in multiple other patientsCH00-090 change found in multiple other patients Identical single base pair change found in affected relative of CH97-042Identical single base pair change found in affected relative of CH97-042

Sulagna Saitta, Brianne O’LoughlinSulagna Saitta, Brianne O’Loughlin

CH97-042 and D96-108 Single b.p. ChangeCH97-042 and D96-108 Single b.p. ChangeCH97-042 and D96-108 Single b.p. ChangeCH97-042 and D96-108 Single b.p. Change

Sulagna Saitta, Brianne O’LoughlinSulagna Saitta, Brianne O’Loughlin

rtPCR of Coding Sequence from CH01-061 Full Length cDNA

rtPCR of Coding Sequence from CH01-061 Full Length cDNA

CH

01-0

61 (

2C

H01

-061

(2

l)l)

CH

01-0

61 (

5C

H01

-061

(5

l)l)

Future PlansFuture Plans

Select more patients based on the zebrafish Select more patients based on the zebrafish phenotype and transgenic mouse phenotypephenotype and transgenic mouse phenotype

Examine differences in expression in CH01-061 Examine differences in expression in CH01-061 as well as patients with point mutationsas well as patients with point mutations

Edg2/LPA1: The ZebrafishEdg2/LPA1: The ZebrafishEdg2/LPA1: The ZebrafishEdg2/LPA1: The Zebrafish Zebrafish edg2/lpa1 cDNA I.M.A.G.E. clone has Zebrafish edg2/lpa1 cDNA I.M.A.G.E. clone has

been sent and used as an been sent and used as an in situin situ probe in E. probe in E. Weinberg’s labWeinberg’s lab

Edg-2 is expressed in early lateral mesoderm and in Edg-2 is expressed in early lateral mesoderm and in the developing heart in zebrafishthe developing heart in zebrafish

Sections and staining to look at morphologic Sections and staining to look at morphologic differencesdifferences

Sulagna Saitta, Eric Weinberg, G. BellipanniSulagna Saitta, Eric Weinberg, G. Bellipanni

EDG-2 and ZebrafishEDG-2 and Zebrafish

EDG-2 localizes to the zebrafish heart and brain at 34 hpfEDG-2 localizes to the zebrafish heart and brain at 34 hpf

G. Bellipanni,G. Bellipanni, PhDPhD

EDG-2 and ZebrafishEDG2/LPA1

EDG-2 and ZebrafishEDG2/LPA1

Edg-2 in both heart chambers at 48 hpfEdg-2 in both heart chambers at 48 hpf

G. Bellipanni, PhDG. Bellipanni, PhD

EDG-2 and ZebrafishEDG-2 and Zebrafish

Morpholino experiments using a single MO to Morpholino experiments using a single MO to

block translation of the Edg-2 protein block translation of the Edg-2 protein

demonstrate an effect on cardiovascular functiondemonstrate an effect on cardiovascular function

Confirmation of specificity of MO by repeating Confirmation of specificity of MO by repeating

with another construct and rescuing phenotype with another construct and rescuing phenotype

with Edg-2 RNAwith Edg-2 RNA

Sulagna Saitta, Eric Weinberg, G. BellipanniSulagna Saitta, Eric Weinberg, G. Bellipanni

EDG2/LPA1EDG2/LPA1

Mouse null mutants generated (Scripps)Mouse null mutants generated (Scripps) Neonatal lethal in 50%; up to 20% fetal loss Neonatal lethal in 50%; up to 20% fetal loss

after E15after E15 Impaired feeding and sizeImpaired feeding and size Increased Schwann cell apoptosisIncreased Schwann cell apoptosis Abnormal peripheral nerve pain responseAbnormal peripheral nerve pain response

Ken is in discussion with Jerold Chun to Ken is in discussion with Jerold Chun to get the mice hereget the mice here

Tissue Culture and DNA CoreTissue Culture and DNA Core

Samples ReceivedSamples Received

Probands = 90Probands = 90 Parents = 136Parents = 136 Others = 6 (siblings, grandparents etc.)Others = 6 (siblings, grandparents etc.)

Total SCCOR samples (‘01-’05) 516 FISHedTotal SCCOR samples (‘01-’05) 516 FISHed DeletedDeleted 19 (3.7%) 19 (3.7%) KaryotypesKaryotypes 411 (0.7%) 411 (0.7%)

TOTAL Samples = 232TOTAL Samples = 232