Transmitted imbalances without phenotypic effect; new examples detected with oligonucleotide array CGH (oaCGH).

1
Transmitted imbalances without phenotypic
effect new examples detected with
oligonucleotide array CGH (oaCGH).
Association for Clinical Cytogenetics
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 8BJ3
Human Genetics DivisionUniversity of Southampton
University School of Medicine, Southampton
2
Chromosomal CNVs
Sub-microscopic CNVs
Iafrate AJ et al, Nat Genet, 36949-510, 2004
200 chromosomal transmitted chromosomal
imbalances in the Chromosome Anomaly
Collection since 1986
gt11,000 sub-microscopic CNVs in the Database of
Genomic Variants since 2004
http//projects.tcag.ca/variation/
www.ngrl.org.uk/Wessex/collection/
3
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
4
Customised Agilent 4x44K array
CoverageMultiple Formats

• High Density, Wide Genome Screening
• 1 x 1 million

• Medium Density
• 2 x 250K
• 4 x 120K

750K array

• Low Density for Multiple Samples
• 8 x 60K

Summer 2007
1,000K array
Summer 2008
8x60K should reduce costs to lt100
5
369 cases processed so far - novel CNVs in
DD/MR/CA group 19/235 8.1
6
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.
(subtel MLPA And FRAXA normal)
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) (B35CHR8128199767-gt13375745
7-)
Chromosome Anomaly Collection (www.ngrl.org.uk/Wes
sex/collection) has one precedent ascertained at
prenatal diagnosis and found in
the phenotypically normal mother (Batanian et al
Clin Genet 200160371-373).
7
Case 1 de novo CNV
KCNQ3 potassium voltage-gated 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 Ommens estimate of 0.14 de novo CNVs
per generation
8
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
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 2002111285-
288)
Brother del(8)(p23.1p23.2) phenotypically normal
Proband del(8)(p23.1p23.3)
46,XX,del(8)(p23.1p23.3)mat.
9
Ensembl v48 gene content With Redon CNVs in
black
Case 2 Chromosomal CNV of 8p23.1-p23.2
Composite array profile of proband, mother and
brother
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
10
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
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)(B35CHR17116,543-gt769,430-)
11
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 200239734-740).
A number of copy number variations of this region
are present in the Database of Genomic
Variants (http//projects.tcag.ca/variation/).
The Transmitted Sub-Telomeric Imbalance
Collection
www.ngrl.org.uk/Wessex/subtel_collection
Conclude non-causal CNV
12
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.
46,XX,?del(1)(q21q21). arr cgh del(1)(q21.1q21.1)(
B35CHR1145031367-gt146201576-). ish
del(1)(533N14-)
13
Cases 4/5/6 Interstitial deletion of 1q21.1 of
uncertain significance
Pro- band Phenotype In- heritance 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) (B35CHR1145031367-gt146201576-)
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) (B35CHR1145031367-gt146201576-)
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) (B35CHR1145031367-gt146201576-
14
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 200780232-240) 2.
International collaboration needed to establish
possible significance.
15
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. Submission to existing databases e.g. 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
16
Acknowledgements
Association for Clinical Cytogenetics
Thank you for your attention
Sanger Institute for 37k cloneset
National Genetics Reference Laboratory
17
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)(B35CHR16732151
18-gt73363910-). ish del(16)(144N1dim,135N16dim)mat
149 kb deletion of 16q22.3
18
Case 4 CNV with causal mutation
RFWD3 and FA2H deleted but
found in phenotypically normal mother
and novel de novo c.3125GgtA (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
19
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
316445-449
Reduction in median copy number of 8p23.1 DEFB4
defensin genes from 4 to 3 confers
predisposition to Chrohns disease Fellermann et
al Am J Hum Genet, 200679439-444
11,784 copy number variants in the Database of
Genomic Variants http//projects.tcag.ca/variation
/ Nov 29 2008 -
Wong et al, Am J Hum Genet 20078091-104
Predict that much of this variation will be
phenotypically silent and some may be
cytogenetically visible
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