1. Detecting Low Level Mosaicism of Trisomy 9 with Microarray Analysis
Shamsa Naqvi, CG
Jennifer Crawford, CG
Jillene Kogan, M.D., Ph.D.
Amanda Fortier, Ph.D.
Debra Rita, M.D.
ACL Laboratories
Rosemont, IL

2. Outline Introduction to Trisomy 9 mosaicism Use of Microarray
Improved analysis techniques
Case studies
Utility of microarray in detecting low levels of mosaicism

3. Background Mosaic trisomy 9 is rare and hard to detect
1973: 47,XY,+9[4]/46,XY[46]
Extra chromosome is lost in culture
Lifespan depends on severity of phenotype and the percentage of mosaicism present
Full trisomy 9 is mostly detected on autopsies of fetal demises
Rarity is mild enough that incidence is unknown
Found in both male and female population
sometimes individuals are not even detected/diagnosed
Longest survival

4. Mosaic Trisomy 9 Phenotypes
Small jaw, low set ears, microcephaly
Bruns D Am J Med Genet Part A 155:1033–1039.
Craniofacial: Micrognathia, low-set ears, microcephaly
Growth: Prenatal growth retardation
Performance: Severe intellectual disability, developmental delay
Skeletal: Joint Contractures
Cardiac: Congenital heart defects
Other: Feeding and respiratory difficulties in the immediate postnatal period

5. Clinical Laboratory Evaluation
Conventional Cytogenetics
Frequently too subtle to be detected
Need many metaphases
FISH
Phenotype not specific
Microarray for better diagnosis
Most specific
Need to maximize the functions
of the software
FISH-phenotype is not specific, must know exactly which probe to order (also need to consider the probability of full vs. partial trisomy)
Microarray-most specific-quantify the level of mosaicism within the patients entire genome

6. ACL Microarray Process
Specimen Type
Postnatal Peripheral Blood
Indications when microarray is ordered:
Developmental delay, autism spectrum disorder, congenital heart defects, dysmorphic features, mental retardation, etc.

7. ACL Workflow Sample Processing
Wet lab preparations (Affymetrix Cytoscan HD)
Intense data analysis by Technologists and Directors
Reported to ordering Physician
Abnormality confirmation by chromosome analysis or FISH as needed

8. ChAS 2.1.0

9. Typical view of ChAS with patient data

10. Typical view of ChAS with patient data
Smooth Signal: Gaussian Smoothed Calibrated Copy Number Estimate
Data type which displays “non-integer copy number changes”- helps to see Mosaicism

11. Detail View: Patient data compared simultaneously against a known normal patient
Loss
Gain
Loss
Gain
Chromosome 14

12. Case 1 Case 2 Case 3 Patient characteristics: HX: One month Female
DX: Dysmorphic facial features
Chromosome Result:
46,XX in 20 cells
Microarray Result:
arr 9p24.3q34.3(36, ,211,203)X2~3 (64%)
Confirmative Result:
Extensive chromosome analysis workup on cultured peripheral blood specimen indicated 14% trisomy 9
Case 2
HX: One month Male
DX: Poor feeding, abnormal cry
47,XY,+mar* in 20 cells
*chromosome marker made of presumably noncoding material was seen
arr[hg 19] 9p24.3q34.3(36, ,211,203)X2~3 (36%)
21.5% in direct FISH preparation
11.5% in cultured cells
Case 3
HX: Two Year old Female
DX: Developmental Delay
46,XX in 20 cells
arr[hg 19] 9p24.3q34.3(36, ,211,203)X2~3 (14%)
10.5% in direct FISH preparation
5.5% in cultured cells

13. Comparison of tracks of 3 cases against a normal chromosome 9
Patient with 64% mosaicism
Patient with 36% mosaicism
Allele peaks- heterozygous lines saparates in 64% mosaicism then less clear in 36% and imperceptible/invisible in 14%
Patient with 14% mosaicism
Chromosome 9

14. Calculating Percentage Mosaicism
(Mean - Copy Number State) x 100 = Percentage Mosaicism
example: (2.14-2) x 100 =14%

15. Same 3 cases: Smooth Signal with Graph Settings as ‘Line’
with Line

16. Magnified Smooth Signal with Graph Settings as ‘Line’
Case 1
Case 2
Case 3
“Smooth Signal”
With Heat Map

17. Other mosaic abnormalities detected using the Heat Map
Mosaic Gain
12p13.33p11.1 (8% by Microarray)
Pallister Killian Syndrome
FISH 1.5% in cultured cells
15q11.2q26.3 (12%)
2% by chromosome analysis
Mosaic Loss
18q21.31-q23 (13%) 46,XX,del(18)(q21.31q23)[3]/46,XX[7]
Mosaic Interstitial Gain
11q21.1-q23.3 (15%)
12% by FISH
% does not represent the degree of mosaicism in other tissues.

18. Conclusion
Microarray reveals cryptic unbalanced genetic material that may not be detected by conventional cytogenetics
Uncultured samples more accurately reflect the percentage of mosaicism than cultured samples
Use of ‘heat map’ function on all of our cases before sign out is an added check to rule out mosaicism

19. References
Bruns D Presenting physical characteristics, medical conditions and developmental status of long-term survivors with trisomy 9 mosaicism. Am J Med Genet Part A 155:1033–1039.
Slavotinek, Anne and Maj Hultén, Unique University of California, San Francisco and University of Warwick, UK Jan 2014.
Blake C. Ballif, et al Detection of Low-Level Mosaicism by Array CGH in Routine Diagnostic Specimens. Am J Med Genet Part A 140A:2757–2767.
Jones, Kenneth Lyons , et al. Smith's Recognizable Patterns of Human Malformation, 5e. Elsevier.1996.Print.
NORD, National Organization for Rare Disorders Chromosome 9, Trisomy Mosaic: Synonyms of Chromosome 9, Trisomy Mosaic. 2 May 2014.
Bruns. D Southern Illinois University, 24 Jan 2014.
Affymetrix® Chromosome Analysis Suite (ChAS) 2.0™ Software User Manual

20. Special Thanks Coauthor of the presentation: Jennifer Crawford CG
Directors: Jillene Kogan, M.D., Ph.D., Amanda Fortier, Ph.D., Debra Rita, M.D.
Supervisor: Ann Marie Rupp MA CG
Microarray Tech Specialist: Aida Catic MA CG
Staff at the ACL Cytogenetics Department
Advocate Health Care

21. Any Questions?

22. Same 3 cases: Smooth Signal with Graph Settings as ‘Heat Map’

23. CASE #2