Transcript Slide 1

Association
for Clinical
Cytogenetics
Transmitted imbalances
without phenotypic effect;
new examples detected with
oligonucleotide array CGH (oaCGH).
John CK Barber [1,2,3], Shuwen Huang [1], Viv K Maloney [1], John A Crolla [1,2,3].
[1] National Genetics Reference Laboratory (Wessex)
[2] Wessex Regional Genetics Laboratory
Salisbury NHS Foundation Trust, Salisbury, SP2 8BJ
[3] Human Genetics Division
University of Southampton University
School of Medicine, Southampton
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Chromosomal CNVs
Sub-microscopic CNVs
Iafrate AJ et al, Nat Genet, 36:949-510, 2004
~ 200 chromosomal transmitted
chromosomal imbalances in the
Chromosome Anomaly Collection
since 1986
www.ngrl.org.uk/Wessex/collection/
>11,000 sub-microscopic CNVs in
the Database of Genomic Variants
since 2004
http://projects.tcag.ca/variation/
How to deal with Copy Number Variations (CNVs):
1. If de novo, causal
2. If transmitted from a phenotypically normal parent,
coincidental.
3. If the number of phenotypically normal CNV carriers is
greater than the number of affected family members,
coincidental.
4. If the number of phenotypically affected CNV carriers
is greater than the number of normal family members,
non-penetrant but causal.
5. If the proportion of CNVs in a population of affected individuals
is significantly greater
than that in the normal population,
predisposition, may be causal.
6. We need to consider how best to share our accumulating experience
of novel copy number variants
with the introduction of diagnostic array CGH
Customised Agilent 4x44K array
Coverage
Multiple Formats
•High Density,
Wide Genome
Screening
•1 x 1 million
•Medium Density
•2 x 250K
•4 x 120K
Summer 2007
750K array
Summer 2008
1,000K array
8x60K should reduce costs to <£100
•Low Density for
Multiple
Samples
•8 x 60K
369 cases processed so far - novel CNVs in DD/MR/CA group – 19/235 = 8.1%
DD/MR/CA - novel CNVs
del(1)(q21.1q21.1) x3
dup(3)(p14.3)pat
dup(4)(q34.3)mat
dup(4)(q13.3)not known
dup(X)(p22.31)mat
del(5)(p14.2)pat
dup(5)(q15q15)pat
dup(12)(q24.23q24.31)mat
del(7)(p12.1->p11.2)mat
del(8)(p23.1p23.3)mat
del(8)(q24.13q24.22)dn
dup(14)(q32.12->q32.13)pat
del(15)(q13.1q13.2)pat
del(16)(p12.1)mat
del(16)(q22.3)mat
del(17)(p13.3p13.3)
dup(17)(q25.1)pat
dup(20)(p11.21)pat
dup(X)(p22.31)mat
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del
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1q21.1 syndrome
STS
Sharp et al 2008
EHTM1 mutation
VCX,STS,VCX-3A
Case 1: de novo CNV
1991: pre-term baby, IVF conception,
infantile spasms, epicanthus,
hypertelorism, prominent tongue.
2005: persistent heterotopias on MRI
and learning disability.
128,199,767
(subtel MLPA
And
FRAXA normal)
133,757,457
Min 5.56 Mb deletion of 8q24.21 to
8q24.22 including 1 novel and 13
known genes
46,XY,del(8)(q24.13q24.22)dn.
ish del(8)(c-myc-).mlpa subtel(PO36Bx2).
arr cgh del(8)(q24.21q24.22)
(B35:CHR8:128199767->133757457-)
Chromosome Anomaly Collection
(www.ngrl.org.uk/Wessex/collection) has one precedent
ascertained at prenatal diagnosis and found in the
phenotypically normal mother
(Batanian et al Clin Genet 2001;60:371-373).
Case 1
de novo CNV
KCNQ3
KCNQ3
potassium voltagegated channel KQT-like
protein
*602232
Expressed in brain;
recessive mutations
cause benign familial
neonatal convulsions
type 2 (BFNC2)
Clinical Genetics team:
infantile spasms/persistent heterotopias
NOT benign familial neonatal convulsions type 2
Conclude:
de novo but non-causal CNV
– note Gert van Ommen’s estimate of 0.14 de novo CNVs per generation
Case 2: Chromosomal CNV of 8p23.1-p23.2:
Girl of 2 referred in 1986 for
developmental delay and failure to thrive
Re-referred in 2006 as a woman of 22 with
moderate learning difficulties,
pulmonary stenosis
and sensorineural deafness.
Father
46,XY
phenotypically
normal
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Mother
del(8)(p23.1p23.2)
phenotypically
normal
Chromosome Anomaly Collection:
Three 2.25 Mb duplication
of 8p23.2 (Harada N et al,
“Duplication of 8p23.2:
a benign cytogenetic
variant?”
Am J Med Genet
2002;111:285-288)
Proband
del(8)(p23.1p23.3)
Brother
del(8)(p23.1p23.2)
phenotypically normal
46,XX,del(8)(p23.1p23.3)mat.
Case 2: Chromosomal CNV of 8p23.1-p23.2:
Composite array profile
of proband, mother and brother
Ensembl v48 – gene content
With Redon CNVs in black
3,176,683
CSMD1
Gene desert
but no
evolutionarily
conserved
non-coding
regions
7,789,937
MCPH1
Variable
defensin
and
olfactory
receptor
cluster
(REPD)
Deletion min 4.61 Mb to max 5.04 Mb
Includes MCPH1 and interrupts the Cub and
Sushi Multiple Domains 1 gene (CSMD1
OMIM 608397) between exons 23 and 27…
…but so do other Copy Number Variations of distal 8p – see Locus 1689 of
the Database of Genomic Variants (http://projects.tcag.ca/variation)!
Conclude: novel non-causal CNV
Case 3: Sub-telomeric CNV of 17p:
Boy of 11 referred with mild developmental delay,
VSD, nasal speech, ?22q11 deletion.
Interstitial deletion of 17p13.3 identified with control probes from the
MLPA sub-telomere kits P023 (GEMIN4) and P070 (RPH3AL) and
confirmed using FISH with BAC RP11-411G7
OaCGH mapped a 653kb interstitial deletion of 17p13.3 distal to the
Miller-Dieker critical region:
A-14-P121738
116,543
GEMIN4
RPH3AL
411G7
A-14-P114056
769,430
46,XY.ish del(17)(p13.3p13.3)(2111b1+,RP11-411G7-,D17S379+)mat.
mlpa 17p13.3(P023 GEMIN4-,P070 RPH3AL-),22q11.2(P023x2).
arr cgh del(17)(B35:CHR17:116,543->769,430-)
Case 3: Sub-telomeric CNV of 17p:
Found in mother
Precedents: two families with
affected probands, unaffected parents
and ~ 600kb sub-telomeric deletions of
17p (Martin et al, J Med Genet
2002;39:734-740).
A number of copy number variations
of this region are present
in the Database of Genomic Variants
(http://projects.tcag.ca/variation/).
Conclude: non-causal CNV
The Transmitted Sub-Telomeric
Imbalance Collection
www.ngrl.org.uk/Wessex/subtel_collection
Cases 4/5/6: Interstitial deletions of 1q21.1 of uncertain significance:
Girl of 1 with mild developmental delay, motor delay, deep set eyes and
flat nasal bridge large big toes, ?Rubinstein-Taybi.
145,031,367
146,201,576
46,XX,?del(1)(q21q21).
arr cgh del(1)(q21.1q21.1)(B35:CHR1:145031367->146201576-).
ish del(1)(533N14-)
Cases 4/5/6: Interstitial deletion of 1q21.1 of uncertain significance:
Proband
Phenotype
Inheritance
Genotype
4.
Development: mild delay, motor delay.
Dysmorphism: deep set eyes and flat nasal
bridge.
Skeletal: large big toes, ?Rubinstein-Taybi.
unknown
arr cgh
del(1)(q21.1q21.1)
(B35:CHR1:145031367>146201576-)
5.
Development : mild global delay, growth
delay, truncal hypotonia
Dysmorphism: mild micrognathia, small mouth
with downturned corners, high palate, bifid
uvula and epicanthic folds; turricephaly.
Skeletal: talipes of R foot at birth.
Other: amputation deformities of left wrist and
foot. Right single palmar crease.
mat
arr cgh
del(1)(q21.1q21.1)
(B35:CHR1:145031367>146201576-)
6.
Development: normal; possible short stature
for family (9th centile).
Dysmorphism: none recorded.
Other: bilateral anterior sutural cataracts at
birth; long-sighted even with replacement
lenses.
unknown
arr cgh
del(1)(q21.1q21.1)
(B35:CHR1:145031367>146201576-
Cases 4/5/6: Interstitial deletions of 1q21.1 of uncertain significance:
Autism Genome Project Consortium – three cases of duplication 1q21.1
seen in patients with autism, one inherited from a normal father
Corresponding deletion and duplication found in other affected
probands and normal parents
Region rich in segmental duplications
May be CNV but not seen in control patients from the HapMap dataset
Conclude:
1. Region distal to the thrombocytopenia-absent radius (TAR) region
(Klopocki et al Am J Hum Genet 2007;80:232-240)
2. International collaboration needed to establish possible significance.
How to deal with Copy Number Variations (CNVs):
Database
Advantages
Disadvantages
1
Publication
Peer reviewed; restricted
access
Very slow
2
“Minimal” publication
eg BMC J Case
Reports
Peer reviewed; open access
Slow
3
Submission to existing databases e.g.:
3a
DECIPHER
Already exists; software
support from Sanger centre
Not curated
3b
ECARUCA
Curated
Would require further software
development
4
Lab specific databases
Data generated under known
conditions
Reduces potential benefit to
others
5
Pan platform
diagnostic
Rapid accumulation of CNV
data
Replicates DOGV on a smaller
scale
6
Platform specific
databases
Consistency of pooled data;
support from commercial
suppliers; part of software
Reduces potential pan-platform
comparisons
7
National//International
control consortium
Data from unaffected
individuals
Cost of identifying and testing
unaffected control cohort
Association
for Clinical
Cytogenetics
Thank you for your attention
Acknowledgements
Sanger Institute for 37k cloneset
National Genetics Reference Laboratory
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Case 4: CNV with causal mutation
Girl of 9 referred with mild to severe developmental delay, hypotonia,
large tongue, facies of del 9q34 syndrome.
Previously 46,XX and negative for PWS/AS, Williams syndrome,
Smith Magenis, Di George, Retts syndrome and mosaic trisomy 21.
46,XX.arr cgh del(16)(q22.3q22.3)(B35:CHR16:73215118->73363910-).
ish del(16)(144N1dim,135N16dim)mat
73,215,118
73,363,910
149 kb deletion
of 16q22.3
Case 4: CNV with causal mutation
RFWD3 and FA2H
deleted but…
found in phenotypically
normal mother…
…and novel de novo c.3125G>A (p.Cys1042Tyr) mutation affecting a conserved
amino acid in the preSET domain of the EHMT1 gene
confirms diagnosis of “9q34 deletion” syndrome in the proband
(Drs HG Yntema and H Scheffer, Nijmegen)
Conclude: novel 16q22.3 CNV
Copy number variation:
•Tiling BAC array shows 3,654
autosomal CNVs in 95 individuals
Genomes of individuals vary by up to
9 Mb or 266 loci (Wong et al, op cit)
•The smaller the probe/target, the
greater the degree of polymorphism
found
Copy number increases in glutamate
signaling genes (GLUR7, CACNG2 and
AKAP5) in patients with bipolar disorder
and schizophrenia - Wilson et al, Hum
Molec Genet, 15, 743-749, 2006
Strong association of de novo
copy number mutations with autism.
Sebat et al, Science 316:445-449
Reduction in median copy number of 8p23.1
DEFB4 defensin genes from 4 to 3
confers predisposition to Chrohn’s disease
Fellermann et al
Am J Hum Genet, 2006;79:439-444
Wong et al, Am J Hum Genet 2007;80:91-104
11,784 copy number variants in the
Database of Genomic Variants
http://projects.tcag.ca/variation/
Nov 29 2008 -
High frequency CNVs to the right in
red (3), green (4), or black (6) individuals;
microRNA (red) and cancer gene CNVs (black) to the left
Predict that much of this variation will be
phenotypically silent and some may be
cytogenetically visible