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 Association for Clinical Cytogenetics

~ 200 chromosomal transmitted chromosomal imbalances in the Chromosome Anomaly Collection since 1986 Chromosomal CNVs >11,000 sub-microscopic CNVs in the Database of Genomic Variants since 2004 Sub-microscopic CNVs Iafrate AJ et al, Nat Genet, 36: , 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 individuals is 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 experience of novel copy number variants with the introduction of diagnostic array CGH

Coverage Multiple 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 K array Summer ,000K array Customised Agilent 4x44K array 8x60K should reduce costs to <£100

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 MRI and learning disability. 128,199, ,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: > ) Min 5.56 Mb deletion of 8q24.21 to 8q24.22 including 1 novel and 13 known genes Chromosome Anomaly Collection ( has one precedentwww.ngrl.org.uk/Wessex/collection ascertained at prenatal diagnosis and found in the phenotypically normal mother (Batanian et al Clin Genet 2001;60: ). (subtel MLPA And FRAXA normal) Case 1: de novo CNV

KCNQ3potassium voltage- gated channel KQT-like protein *602232Expressed 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 1 de novo CNV

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. Mother 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 duplication of 8p23.2 (Harada N et al, “Duplication of 8p23.2: a benign cytogenetic variant?” Am J Med Genet 2002;111: ) Chromosome Anomaly Collection: 46,XX,del(8)(p23.1p23.3)mat.

Composite array profile of proband, mother and brother 3,176,683 7,789,937 Ensembl v48 – gene content With Redon CNVs in black Deletion min 4.61 Mb to max 5.04 Mb CSMD1 MCPH1 Variable defensin and olfactory receptor cluster (REPD) Gene desert but no evolutionarily conserved non-coding regions Includes MCPH1 and interrupts the Cub and Sushi Multiple Domains 1 gene (CSMD1 OMIM ) between exons 23 and 27… …but so do other Copy Number Variations of distal 8p – see Locus 1689 of the Database of Genomic Variants ( 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 the MLPA sub-telomere kits P023 (GEMIN4) and P070 (RPH3AL) and confirmed using FISH with BAC RP11-411G7 A-14-P ,543 A-14-P ,430 OaCGH mapped a 653kb interstitial deletion of 17p13.3 distal to the Miller-Dieker critical region: RPH3AL GEMIN4 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 with affected probands, unaffected parents and ~ 600kb sub-telomeric deletions of 17p (Martin et al, J Med Genet 2002;39: ). A number of copy number variations of this region are present in the Database of Genomic Variants ( The Transmitted Sub-Telomeric Imbalance Collection Conclude: non-causal CNV Found in mother

145,031, ,201,576 46,XX,?del(1)(q21q21). arr cgh del(1)(q21.1q21.1)(B35:CHR1: > ). 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 and flat nasal bridge large big toes, ?Rubinstein-Taybi.

Pro- band PhenotypeIn- heritance Genotype 4.Development: mild delay, motor delay. Dysmorphism: deep set eyes and flat nasal bridge. Skeletal: large big toes, ?Rubinstein-Taybi. unknownarr cgh del(1)(q21.1q21.1) (B35:CHR1: > ) 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. matarr cgh del(1)(q21.1q21.1) (B35:CHR1: > ) 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. unknownarr cgh del(1)(q21.1q21.1) (B35:CHR1: > 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: ) 2. International collaboration needed to establish possible significance.

DatabaseAdvantagesDisadvantages 1PublicationPeer reviewed; restricted access Very slow 2“Minimal” publication eg BMC J Case Reports Peer reviewed; open accessSlow 3Submission to existing databases e.g.: 3aDECIPHERAlready exists; software support from Sanger centre Not curated 3bECARUCACuratedWould require further software development 4Lab specific databasesData generated under known conditions Reduces potential benefit to others 5Pan platform diagnostic Rapid accumulation of CNV data Replicates DOGV on a smaller scale 6Platform specific databases Consistency of pooled data; support from commercial suppliers; part of software Reduces potential pan-platform comparisons 7National//International control consortium Data from unaffected individuals Cost of identifying and testing unaffected control cohort How to deal with Copy Number Variations (CNVs):

Association for 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 ,XX.arr cgh del(16)(q22.3q22.3)(B35:CHR16: > ). ish del(16)(144N1dim,135N16dim)mat Case 4: CNV with causal mutation 149 kb deletion of 16q ,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 FA2H deleted but…

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 Wong et al, Am J Hum Genet 2007;80: The smaller the probe/target, the greater the degree of polymorphism found 11,784 copy number variants in the Database of Genomic Variants Nov 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) 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, , 2006 Predict that much of this variation will be phenotypically silent and some may be cytogenetically visible Strong association of de novo copy number mutations with autism. Sebat et al, Science 316: Copy number variation: 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: