John CK Barber [1,2,3], Shuwen Huang [1], Viv K Maloney [1], John A Crolla [1,2,3].

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 8BJ[3] Human Genetics DivisionUniversity of Southampton University School of Medicine, Southampton

2. 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 >11,000 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): • 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 <£100

5. 369 cases processed so far - novel CNVs in DD/MR/CA group – 19/235 = 8.1%

6. 128,199,767 133,757,457 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) (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).

7. KCNQ3 Case 1 de novo CNV 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

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 2002;111:285-288) Brother del(8)(p23.1p23.2) phenotypically normal Proband del(8)(p23.1p23.3) 46,XX,del(8)(p23.1p23.3)mat.

9. 3,176,683 CSMD1 MCPH1 Variable defensin and olfactory receptor cluster (REPD) Gene desert but no evolutionarily conserved non-coding regions 7,789,937 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. A-14-P121738 116,543 A-14-P114056 769,430 GEMIN4 RPH3AL 411G7 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)(B35:CHR17:116,543->769,430-)

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 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/). The Transmitted Sub-Telomeric Imbalance Collection www.ngrl.org.uk/Wessex/subtel_collection Conclude: non-causal CNV

12. 145,031,367 146,201,576 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)(B35:CHR1:145031367->146201576-). ish del(1)(533N14-)

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

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 2007;80:232-240) 2. International collaboration needed to establish possible significance.

15. How to deal with Copy Number Variations (CNVs):

16. Acknowledgements Association for Clinical Cytogenetics Thank you for your attention Sanger Institute for 37k cloneset National Genetics Reference Laboratory

17. 73,215,118 73,363,910 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 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.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

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 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 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 2007;80:91-104 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