Current Applications for Genomic Microarray Jillene Kogan, M.D., Ph.D. ACL Laboratories Cytogenetics Department July 28, 2016

Objectives 1. Define genomic microarray 2. Review what microarray can and cannot do 3. Discuss applications of constitutional genomic microarray 4. Case examples

What is Microarray? The word “Microarray” can refer to several different things which all involve DNA immobilized on a surface used to analyze multiple genomic fragments at once. Those currently used in cytogenetics to assess copy number are “Genomic microarrays”

Genomic Microarray A technology consisting of DNA immobilized on a surface used to analyze genomic fragments in a high- throughput, parallel assay Can investigate the entire genome at a very high resolution in an unbiased manner Two main types for copy number analysis: Comparative Genomic Hybridization (CGH) Single Nucleotide Polymorphism (SNP)-based microarray

Clinical Indications for Microarray Postnatal Intellectual Disability/Developmental Delay Autism Dysmorphic features Congenital anomalies Prenatal Fetal anomalies Stillbirth Maternal anxiety Both pre- and post-natal Any suspicion for an unbalanced chromosome abnormality Ex. Family history of a predisposing chromosome abnormality Further characterization of a known chromosomal abnormality

History of CGH Early 1990’s metaphase CGH is introduced Test DNA (green) and normal reference DNA (red) is mixed and hybridized to normal metaphase Resolution approximately 5–10 Mb A. 8q24 myc amplification B. gain of 2p21 and 2q21 Test DNA: 45,X Reference DNA: 46,XX Metaphase: 46,XY Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D. 1992. Science. 258: 818-821

Array CGH Introduced in the late 1990’s DNA targets are fixed to a solid support Allows higher resolution depending on the size of the features Types of probes BACS 150-200 Kb Oligos 25-60 bases

BACS and oligos higher resolution

AAGGCTAA --> ATGGCTAA SNP-based microarray Arrays can be more than 2 million probes across the whole genome 25-mer or 50-mer oligonucleotide probes based on SNPs (single nucleotide polymorphisms) SNPs: DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered and is present in at least 1% of the population. AAGGCTAA --> ATGGCTAA

SNP-based microarray Originally designed for whole-genome association studies Can give genotyping data as well as chromosome anomalies Allows detection of polyploidy and homozgosity High density High throughput

Affymetrix

Affymetrix assay

Fluorescence intensity SNP-based microarray Whole genomes are analyzed, but instead of looking at the specific genotypes, we look at gains or losses of DNA For every SNP, we expect to have 2 alleles, which may be homozygous (AA, BB) or heterozygous (AB). Genotyping data Fluorescence intensity Normal Chromosome 12

SNP-based microarray Can detect: Cannot detect: Losses (deletions) Gains (duplications, triplications, etc) Unbalanced rearrangements Mosaicism down to about 20% Copy number variants (CNVs) Runs of homozygosity Polyploidy Cannot detect: Balanced rearrangements *chromosome structural information* Mosaicism under about 20% Changes in genomic regions not represented on the array

or The Limitations of Microarray for Structural Analysis

Pathogenic vs. Benign Everyone has copy number changes Polymorphic copy number variants (CNVs) are considered benign How do you know if a particular deletion or duplication is causative or benign? It is not always clear

CNV vs. Pathogenic More likely pathogenic if: More likely CNV if: Smaller size Duplication Inherited from a normal parent Lower gene content Commonly reported region in database of genomic variants More likely pathogenic if: Larger size Deletion De novo Higher gene content Known microdeletion or microduplication syndrome region

Whole Genome and Segments View

Deletion Example

Duplication Example

Trisomy Example

Trisomy Example

Homozygosity Example

Clinical significance of runs of homozygosity Imprinting conditions Uniparental disomy (UPD) Prader-Willi syndrome, Angelman syndrome, Beckwith Wiedemann syndrome Consanguinity The amount of homozygosity is correlated with degree of parental relatedness Loss of heterozygosity (LOH) in cancer

What can be run on microarray for constitutional analysis? These can be direct or cultured samples Blood Skin fibroblasts Buccal swab (in other labs) Amniotic fluid (currently goes to ARUP) CVS (currently goes to ARUP) POC Fetal tissue, placenta, membrane, cord Maternal cell contamination testing is recommended

Postnatal microarray utility Currently standard of care for idiopathic developmental delay, intellectual disability, autism, or multiple congenital anomalies Diagnostic yield is higher as compared to G-banded karyotyping 10-20% (vs 3%) Autism presents challenges due to “susceptibility” regions Particularly challenging in the prenatal setting

Prenatal microarray utility The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine “…fetus with one or more major structural abnormalities… chromosomal microarray analysis is recommended. This test replaces the need for fetal karyotype.” “…intrauterine fetal demise or stillbirth… chromosomal microarray analysis on fetal tissue… is recommended because of its increased likelihood of obtaining results and improved detection of causative abnormalities.” “Comprehensive patient pretest and posttest genetic counseling from qualified personnel such as a genetic counselor or geneticist regarding the benefits, limitations, and results of chromosomal microarray analysis is essential.” The use of chromosomal microarray analysis in prenatal diagnosis. Committee Opinion No. 581. American College of Obstetricians and Gynecologists. Obstet Gynecol 2013;122:1374–7 Wapner, et al., N Engl J Med. 2012 Dec 6; 367(23): 2175–2184.

Value of genomic microarray in stillbirth Karyotype vs microarray Reddy, et al., N Engl J Med 2012; 367:2185-2193

Value of genomic microarray in stillbirth Conclusions Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained Reddy, et al., N Engl J Med 2012; 367:2185-2193

POC case 1 Chromosome analysis on cultured placental tissue was 46,XX Microarray was performed on frozen direct villi

POC case 1 Microarray analysis revealed 47,XY,+16

POC case 2 Microarray analysis on direct membrane tissue, no chromosomes ordered

POC case 2 All autosomal chromosomes showed 4 allele tracks, suggestive of triploidy

POC case 2 All autosomal chromosomes showed 4 allele tracks, suggestive of triploidy Karyotype: arr (1-22)x3,(X)x2,(Y)x1 Equivalent to 69,XXY

Postnatal case with unexpected findings 5 year old boy with developmental delay 2 findings

Postnatal case with unexpected findings 15q13.3 duplication involving the CHRNA7 gene Encodes nicotinic acetylcholine receptor Common autism/DD/ID susceptibility region, often inherited

Postnatal case with unexpected findings Also detected 7p22.1 small deletion

Postnatal case with unexpected findings Deletion involves the PMS2 gene Associated with Lynch syndrome

Take home points Microarray analysis is very useful for high resolution copy number analysis in the prenatal and postnatal setting Cannot directly ascertain chromosome structure Recommended as a first line test for children with DD/ID, autism, and multiple congenital anomalies Recommended for prenatal cases and stillbirths with anomalies Sometimes presents uncertain or unexpected results

Thank you! Questions?