Chromosomal Microarray Analysis (CMA) Medical Genetics Laboratories Department of Molecular and Human Genetics Baylor College of Medicine Table of Contents • Overview of CMA • Examples of Common Findings • Examples of Mosaicism • Examples of Complex Abnormalities • Examples of Small Copy Number Variants • CMA Comprehensive-CMA plus SNPS • Resolving Variants of Uncertain Significance • Prenatal CMA Considerations • Prenatal CMA Case Examples • Types of Cancer Arrays • Conclusion Overview of CMA Chromosomal Microarray Analysis • CMA is an array-based comparative genomic hybridization methodology that allows for analysis of the chromosomes for a large number of genetic disorders. • With a single test, CMA can identify the abnormalities that are detectable by both routine chromosome analysis and FISH analysis. • CMA has greater sensitivity than older methods of chromosome analysis. Genomic Resolution Karyotype [4-5 Mb, whole genome] FISH [40 to 250 kb per probe, single site] CMA [average resolution ~30kb, whole genome] 10 Limits of Resolution Chromosome vs. Array CGH Analysis Chromosome 1 4 Mb region of microarray data Limits of detection for G-banded showing a 1 Mb deletion chromosome analysis is 4-5 Mb encompassing ~70 oligos What is included? • Whole genome copy number analysis • Coverage is more dense at genomic sites associated with known genetic conditions – Currently 180,000 to 400,000 probes (oligos) covering the genome (depending on version) – Exon coverage of 1700-4400 disease-associated genes (depending on version) • Pericentromeric regions – useful for detecting marker chromosomes • Subtelomeric regions • Backbone coverage (30kb resolution) • Verification of results by FISH analysis and/or partial karyotype when indicated • Parental studies to determine if observed copy number changes are inherited or de novo CMA Process [A] Experimental Procedure [B] Laser Scanner [D] Array Profile Patient Control Mix Hybridization of genomic DNA to the array of small DNA fragments [C] Actual Array (oligo probes) Laser Scanner Duplication Deletion Shinawi, M. and Cheung, S.W. (2008) The array CGH and its clinical applications. Drug discovery today, 13, 760-70. 1. Evolution of CMA - increasing pixels! Feb 2004 July 2005 Nov 2006 Mar 2007 Feb 2008 June 2009 VERSION V4 BAC V5 BAC V6 BAC V6 OLIGO V7 OLIGO V8 OLIGO 180K Interrogating probes 366 BACs 853 BACs 1475 BACs 44K oligos 105K oligos oligos 420 genes >1700 genes Genomic disorders 40 75 >140 >140 (+61 regions *) (exons) Subtelomeric regions 41 41 41 41 41 41 # of clones / subtel ~ 4 ~10 ~10 ~10 (x20) NA NA Coverage per subtel ~ 5 Mb ~ 10 Mb ~ 10 Mb ~ 10 Mb NA NA Pericentromeric regions none 43 43 43 43 43 # clone / region ~3 ~ 3-5 ~ 3-5 ~3-5 (x20) NA NA 1 clone per 1 clone per Backbone coverage NA NA 1 band 1 band 30 kb 30 kb LCR regions 290 290 More More More More Disorders Design Disorders disorders BAC clones Disorders mito FISH test + subtel + each chr oligo + mito + exon Improvement + subtel + pericentr band emulation + LCR regions coverage Detection Rate 6.50% 9.04% ~12% ~12.5% ~15.4% TBD Evolution of CMA - increasing pixels! N >46,000 Whole Genome Coverage June 2009 Oct 2010 July 2011 July 2011 2012 V8.0 OLIGO V 8.3 OLIGO V9.0 OLIGO VERSION V8 OLIGO +SNP Screen V 8.1.1 OLIGO +SNP Screen +SNP Screen Interrogating 180K Oligos 400K Oligos 180K Oligos 400K Oligos 400K Oligos probes >1700 genes >1700 genes 1779 genes 1936 genes 4864 genes Genomic disorders Exon coverage Exon coverage Exon coverage Exon coverage Exon coverage HG18 HG18 HG19 HG19 HG19 Subtelomeric regions 41 41 41 41 41 Pericentromeric regions 43 43 43 43 43 Backbone coverage 30 kb 30 kb 30 kb 30 kb 30 kb LCR regions 290 290 290 290 290 More Disorders More Disorders More Disorders More Disorders More Disorders Mito Mito Mito Mito Mito + exon + exon coverage + exon coverage + exon coverage + exon coverage + Design coverage + Additional 79 Additional 200 Additional 2500 Improvement 120K SNP genes genes +120K SNP genes +120K SNP 38 non-coding regulatory elements Advantages of CMA • Screens for a large number of disorders simultaneously • Detects conditions that are difficult to identify clinically – Atypical or mild phenotypes; e.g., VCFS/DiGeorge – Conditions that lack distinctive features • Detects deletions and duplications simultaneously • Detects submicroscopic unbalanced chromosome rearrangements • Detects mosaicism (as low as 10%) Cheung et al. (2007) Am J Med Genet A. (15):1679-86 • Detects interstitial subtelomeric deletions/ duplications Limitations of CMA • Does not detect balanced translocations, inversions, low level mosaicism, point mutations • Can detect copy number variants (CNVs) of unknown clinical significance – Most are easily resolved by parental studies, however, clinicians should carefully evaluate parental phenotypes and developmental histories so data can be appropriately interpreted. – CNVs <500 kb containing no identified genes at time of analysis are not reported – CNVs >300 kb containing genes, even if clinical significance is unknown, are reported – CNVs <300 kb must contain a gene known to be associated with disease in order to be reported Examples of Common Findings Normal Result Trisomy 21 Chromosome 21-specific plot DiGeorge Syndrome / VCFS Top: Whole genome view of CMA data Bottom: • Left: FISH confirmation • Inset: Partial karyotype of chromosome 22 (arrow points to deleted 22) • Right: Chromosome 22-specific oligo plot of CMA data. DiGeorge Syndrome / VCFS del 22q11.2 Microduplication 22q11.2 Syndrome (3 Mb ) • Duplication of the DiGeorge / Velocardiofacial Syndrome region on chromosome 22q11.2 Microduplication 22q11.2 Syndrome (3 Mb ) Chromosome 22 Partial karyotype of chromosome 22 (arrow indicates duplicated 22) FISH confirmation 3 red signals indicates duplication Example of Mosaicism Mosaicism Example Indication – Microcephaly, congenital vertical talus arr 18q21.2q23(47898780-76103255)x1.nuc ish 18q21.2(RP11- 25O3x1)[45/200]dn Chromosomal Microarray Analysis revealed an approximately 28.2 Mb LOSS in copy number in the distal and subtelomeric regions of the long arm of chromosome 18 suggestive of mosaicism. This deletion includes the critical region of chromosome 18q deletion syndrome (OMIM 601808). FISH analysis and partial chromosome analysis revealed mosaicism for a deletion in the long arm of one chromosome 18 in 22% (45/200) of interphase cells examined. The remaining 78% (155/200) of cells showed a normal hybridization pattern. UPDATE: Parental FISH analysis with the above clone showed no evidence of the same LOSS in the father (KCL144199). UPDATE: Parental FISH analysis with the above clone showed no evidence of the same LOSS in the mother (KCL 146866). Therefore, this result most likely represents a de novo event. Genetic counseling is warranted. Mosaicism (contin’d) Indication – Microcephaly, congenital vertical talus Examples of Complex Abnormalities Complex X Chromosome Abnormality Complex X Chromosome Abnormality Chromosome X-specific plot Complex Chromosome 1 Abnormality A Chr 1 BCM array B 1q41q42 microdeletion 3.1 Mb 4.4 Mb TAR region syndrome region (dup) (dup) Nimblegen duplication not duplicated 1q42.12 1q42.3q43 221,378,468 232,592,278 2.1 224,471,629 236,988,147 0.3 Mb 4.7 Mb (del) nml van der Woude 1.8 Mb (del) 1q32.2 del 1q43 205,060,076 AKT3 237,337,843 209,804,762 not deleted 239,105,025 D E F G C RP11-279E18 RP11-339I11 RP11-478H16 RP11-478H16 D1Z1 D1Z1 RP5-1090A23 RP5-1090A23 Complex Chromosome 1 Abnormality A. CMA identified a complex rearrangement including two gains and two losses in chromosome 1q. B. High density array CGH using Nimblegen 2.1M array showed a 0.3 Mb single copy sequence between the distal duplication and deletion. C. Chromosome analysis showed that the gained materials (red arrow) due to the duplications were translocated into the 1q32 region. D-G. FISH analyses confirmed the deletions in 1q32.2 (D) and 1q43 (E) and the two copy number gains (F). In addition, FISH analysis on metaphase cells (G) suggested an inversion between the two regions with copy number gains and showed the duplicated segments of 1q42.12 and 1q42.3q43 were located next to each other as indicated by a white arrow. Liu et al. Cell In Press Evaluation for a Marker chromosome Evaluation for a Marker Chromosome Chromosome 1-specific plot CMA identified that the marker marker chromosome originated from chromosome 1. Examples of Small Copy Number Variants (including exon deletions) Deletion of an Exon of ERBB4 on 2q34 INTERPRETATION OF RESULTS: Chromosomal Microarray Analysis revealed a LOSS in copy number in the distal long arm of chromosome 2, spanning a minimum of 0.229 Mb and a maximum of 0.296 Mb. This deletion disrupts the ERBB4 (erythroblastic leukemia viral oncogene homolog 4) gene. A recent publication describes haploinsufficiency of ERBB4 gene in a patient with early myoclonic encephalopathy and profound psychomotor delay (Eur J Hum Genetics. 2009 Mar 17(3):378-82). Clinical correlation is recommended and genetic counseling is warranted. Deletion of an Exon of ERBB4 on 2q34 Whole chromosome 2 Deletion of Exons of EP300 on 22q13.32 • Figure 3 A. Profile of the microarray analysis showing the deleted region as indicated in the red circle [the gain on Xp as shown in green dots is also present in the mother (data not shown)]; • B. The deleted oligos displayed in the UCSC genome browser corresponds to the exons of the CREBBP gene. • C. The MLPA profile demonstrated copy number changes in the 2 exons of the CREBBP gene (exons 27- 28). • D. The deletion profile