Transmitted imbalances without phenotypic effect;

new examples detected with oligonucleotide array CGH (oaCGH).

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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

Associationfor Clinical

Cytogenetics

www.ngrl.org.uk/Wessex/collection/

~ 200 chromosomal transmitted chromosomal imbalances in the Chromosome Anomaly Collectionsince 1986

Chromosomal CNVs

>11,000 sub-microscopic CNVs in the Database of Genomic Variantssince 2004

Sub-microscopic CNVs

http://projects.tcag.ca/variation/

Iafrate AJ et al, Nat Genet, 36:949-510, 2004

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.

5. If the proportion of CNVs in a population of affected individualsis significantly greater

than that in the normal population,predisposition, may be causal.

4. If the number of phenotypically affected CNV carriers is greater than the number of normal family members,

non-penetrant but causal.

6. We need to consider how best to share our accumulating experienceof novel copy number variants

with the introduction of diagnostic array CGH

CoverageMultiple Formats

•Low Density for Multiple Samples

•8 x 60K

•Medium Density

•2 x 250K

•4 x 120K

•High Density, Wide Genome Screening

•1 x 1 million

Summer 2007 750K array

Summer 2008 1,000K array

Customised Agilent 4x44K array

8x60K should reduce costs to <£100

DD/MR/CA - novel CNVsdel(1)(q21.1q21.1) x3 1 del 1.16 Mb 1q21.1 syndromedup(3)(p14.3)pat 3 dup 1.12 Mbdup(4)(q34.3)mat 4 dup 2.05 Mbdup(4)(q13.3)not known 4 dup 1.3 Mbdup(X)(p22.31)mat X dup 1.0 Mb STSdel(5)(p14.2)pat 5 del 0.5 Mbdup(5)(q15q15)pat 5 dup 4.31 Mbdup(12)(q24.23q24.31)mat 12 dup 3.46 Mbdel(7)(p12.1->p11.2)mat 7 del 5.35 Mbdel(8)(p23.1p23.3)mat 8 del 4.61 Mbdel(8)(q24.13q24.22)dn 8 del 5.56 Mbdup(14)(q32.12->q32.13)pat 14 dup 0.25 Mbdel(15)(q13.1q13.2)pat 15 del 1.41 Mb Sharp et al 2008del(16)(p12.1)mat 16 del 0.8 Mbdel(16)(q22.3)mat 16 del 0.2 Mb EHTM1 mutationdel(17)(p13.3p13.3) 17 del 0.6 Mbdup(17)(q25.1)pat 17 dup 0.8 Mbdup(20)(p11.21)pat 20 dup 0.3 Mbdup(X)(p22.31)mat X dup 1.6 Mb VCX,STS,VCX-3A

369 cases processed so far - novel CNVs in DD/MR/CA group – 19/235 = 8.1%

1991: pre-term baby, IVF conception,infantile spasms, epicanthus,hypertelorism, prominent tongue. 2005: persistent heterotopias on MRIand learning disability.

128,199,767

133,757,457

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-)

Min 5.56 Mb deletion of 8q24.21 to 8q24.22 including 1 novel and 13 known genes

Chromosome Anomaly Collection(www.ngrl.org.uk/Wessex/collection) has one precedentascertained at prenatal diagnosis and found in thephenotypically normal mother(Batanian et al Clin Genet 2001;60:371-373).

(subtel MLPAAnd

FRAXA normal)

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)

KCNQ3

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 1de novo CNV

Case 2: Chromosomal CNV of 8p23.1-p23.2:

Girl of 2 referred in 1986 for developmental delay and failure to thriveRe-referred in 2006 as a woman of 22 with

moderate learning difficulties, pulmonary stenosis

and sensorineural deafness.

NMother

del(8)(p23.1p23.2)phenotypically

normal

Proband del(8)(p23.1p23.3)

Brother del(8)(p23.1p23.2)

phenotypically normal

Father 46,XY

phenotypically normal

Three 2.25 Mb duplicationof 8p23.2 (Harada N et al,“Duplication of 8p23.2:a benign cytogeneticvariant?”Am J Med Genet2002;111:285-288)

Chromosome Anomaly Collection:

46,XX,del(8)(p23.1p23.3)mat.

Composite array profileof proband, mother and brother

3,176,683

7,789,937

Ensembl v48 – gene contentWith Redon CNVs in black

Deletion min 4.61 Mb to max 5.04 Mb

CSMD1 MCPH1

Variabledefensin

and olfactory receptor cluster (REPD)

Gene desert but no

evolutionarily conservednon-coding

regions

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)!

Case 2: Chromosomal CNV of 8p23.1-p23.2:

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 theMLPA sub-telomere kits P023 (GEMIN4) and P070 (RPH3AL) andconfirmed using FISH with BAC RP11-411G7

A-14-P121738116,543

A-14-P114056769,430

OaCGH mapped a 653kb interstitial deletion of 17p13.3 distal to the Miller-Dieker critical region:

RPH3ALGEMIN4

411G7

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:

Precedents: two families withaffected probands, unaffected parentsand ~ 600kb sub-telomeric deletions of 17p (Martin et al, J Med Genet2002;39:734-740).

A number of copy number variationsof this region are presentin the Database of Genomic Variants(http://projects.tcag.ca/variation/).

www.ngrl.org.uk/Wessex/subtel_collection

The Transmitted Sub-Telomeric Imbalance Collection

Conclude: non-causal CNV

Found in mother

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 deletions of 1q21.1 of uncertain significance:

Girl of 1 with mild developmental delay, motor delay, deep set eyes andflat nasal bridge large big toes, ?Rubinstein-Taybi.

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)(B35:CHR1:145031367->146201576-)

5. Development : mild global delay, growth delay, truncal hypotoniaDysmorphism: 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 deletion 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

May be CNV but not seen in control patients from the HapMap dataset

Region rich in segmental duplications

Cases 4/5/6: Interstitial deletions of 1q21.1 of uncertain significance:

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.

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 specificdatabases

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

How to deal with Copy Number Variations (CNVs):

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Associationfor Clinical

Cytogenetics

Thank you for your attention

Sanger Institute for 37k cloneset

National Genetics Reference Laboratory

Acknowledgements

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

Case 4: CNV with causal mutation

149 kb deletion of 16q22.3

73,215,118

73,363,910

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

Case 4: CNV with causal mutation

RFWD3 and FA2Hdeleted but…

High frequency CNVs to the right inred (3), green (4), or black (6) individuals;microRNA (red) and cancer gene CNVs (black) to the left

Wong et al, Am J Hum Genet 2007;80:91-104

•The smaller the probe/target, the greater the degree of polymorphism found

11,784 copy number variants in the Database of Genomic Variants http://projects.tcag.ca/variation/ Nov 29 2008 -

•Tiling BAC array shows 3,654autosomal CNVs in 95 individualsGenomes of individuals vary by up to 9 Mb or 266 loci (Wong et al, op cit)

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

Predict that much of this variation will be phenotypically silent and some may be cytogenetically visible

Strong association of de novocopy number mutations with autism.Sebat et al, Science 316:445-449

Copy number variation:

Reduction in median copy number of 8p23.1 DEFB4 defensin genes from 4 to 3 confers predisposition to Chrohn’s diseaseFellermann et alAm J Hum Genet, 2006;79:439-444