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

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

3. 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”

4. 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

5. 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

6. 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 Science. 258:

7. 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 Kb
Oligos
25-60 bases

8. BACS and oligos
higher resolution

9. 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

10. 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

11. Affymetrix

12. Affymetrix assay

13. 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

14. 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

15. or The Limitations of Microarray for Structural Analysis

16. 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

17. 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

18. Whole Genome and Segments View

19. Deletion Example

20. Duplication Example

21. Trisomy Example

22. Trisomy Example

23. Homozygosity Example

24. 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

25. 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

26. 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

27. 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 American College of Obstetricians and Gynecologists. Obstet Gynecol 2013;122:1374–7
Wapner, et al., N Engl J Med Dec 6; 367(23): 2175–2184.

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

29. 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:

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

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

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

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

34. 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

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

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

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

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

39. 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

40. Thank you!
Questions?