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
n
lung live
r
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
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Adu
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Rig
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entr
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Lef
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Dex
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Sin
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Inte
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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
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