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What’s Next in Cytogenetics
Molecular Characterization of De Novo Apparently
Balanced Chromosomal Rearrangements to Assess
Pathogenicity by Whole Genome Mate Pair Sequencing
Cherisse A. Marcou, PhD
2016 Cancer Genetics Consortium Meeting
August 9, 2016
©2016 MFMER | slide-1
Apparently balanced chromosomal
rearrangements
• Approximately 1:200 individuals carry an
“apparently balanced” rearrangement
• Is commonly not thought to contribute to an
abnormal phenotype
• Represents an interpretation and counseling
challenge in the setting of an abnormal phenotype
©2016 MFMER | slide-2
Potential pathogenic mechanisms
• Intragenic break resulting in gene disruption of a
dosage sensitive gene
• Dysregulation of a gene or genes distant from
the break due to a position effect
• Cryptic imbalances at or near the breakpoint(s)
• Production of a chimeric gene
• Disruption of conserved non-coding genomic
elements
©2016 MFMER | slide-3
• 10-year study of 377,357 reported
amniocenteses
• Risk of a serious congenital anomaly was:
• 6.1% for de novo reciprocal translocations
• 9.4% for inversions
• 6.7% combined (reciprocal translocations
and inversions)
©2016 MFMER | slide-4
What’s Next??
Next generation sequencing
techniques that address questions that
have historically been answered by
classical cytogenetic methods
©2016 MFMER | slide-5
Study design
• Retrospective review of clinical cases that had
an apparently balanced rearrangement by Gbanding chromosome analysis reported from
the Mayo Clinic Cytogenetics laboratory
• Selected 14 clinical cases to characterize
*Ascertainment bias
• Chromosomal microarray analysis was
performed to identify any cryptic imbalances
• Mate pair sequencing analysis with PCR and
Sanger sequencing characterization of the
breakpoints
©2016 MFMER | slide-6
Mate Pair Sequencing for Rearrangements
Fragment / Biotinylate:
Circularize:
B
B
2-5 kb fragments
Re-Fragment:
~500bp
Capture &
Sequence:
100bp
B
100bp
B
B
B
B
B
By circularizing a longer fragment, enriching for these
fragments, and performing paired end sequencing, the
likelihood of capturing a structural rearrangement event is
significantly increased
Slide modified from Sarah Johnson
©2016 MFMER | slide-7
Bridged coverage:
Slide modified from Sarah Johnson
large fragments more likely to span breakpoint
bridged coverage 5x
*
*
*
Mate pair sequencing does
not provide a
*
15 Paired-End
precise breakpoint location in gDNA. However,
500 bp fragments
the longer fragments and bridged coverage
chr B
chr A
provide genomic sequence*close to the
*
* map the
breakpoint
that
can
be
used
to
15 Mate-Pair
*
*
3000 bp fragments
*
breakpoint
*
*
*
bridged coverage 13x
*
*
*
*
*
WGMP Balanced Rearrangement
Characterization Cohort (n=14)
©2016 MFMER | slide-9
Case 1
• Clinical History
• 1 day old female
• Prenatal diagnosis of a de novo
translocation
• Small for gestational age
• Absent corpus callosum
• Karyotype:
• Array: normal
©2016 MFMER | slide-10
Case 1
46,XX,t(1;9)(q44;p13.2)dn
• Mate Pair Sequencing
Right of
Red
Left of
Blue
©2016 MFMER | slide-11
Case 1
46,XX,t(1;9)(q44;p13.2)dn
• PCR confirmation
Mapped breakpoints to a 5bp region of microhomology
©2016 MFMER | slide-12
Case 1
46,XX,t(1;9)(q44;p13.2)dn
• UCSC browser
• 1q44: AKT3 gene
Intron 1
Disruptions in the AKT3 gene are
associated with agenesis of the corpus
callosum and microcephaly
• 9p13.2: No annotated genes
LOC100506710
~25kb distal
ZCCHC7
~5kb proximal
©2016 MFMER | slide-13
Case 2
• Clinical History
• 6 day old female
• Abnormal facial features
• Abnormal hair patterns
• Passed away on day 13
• Karyotype:
• Array: nml
©2016 MFMER | slide-14
Case 2
46,XX,t(2;3)(q24.1;q28)dn
• Mate Pair Sequencing
©2016 MFMER | slide-15
Case 2
46,XX,t(2;3)(q24.1;q28)dn
• PCR confirmation
Mapped breakpoints to a 4bp region of microhomology
©2016 MFMER | slide-16
Case 2
46,XX,t(2;3)(q24.1;q28)dn
• UCSC browser
• 2q24.1: No annotated genes
Disruptions in TP63 are associated with
multiple syndromes involving abnormal
• 3q28: TP63
gene
facial features and hair
Intron 3
©2016 MFMER | slide-17
WGMP Balanced Rearrangement
Characterization Results (n=14)
©2016 MFMER | slide-18
Summary of WGMP Characterization
Results
• 3/14 (21.4%) had disruption of a known
pathogenic gene
• 6/14 (42.9%) had disruption of at least one
gene of uncertain significance
• 5/14 (35.7%) had breakpoints in intergenic
regions on both chromosomes
We now have a methodology to molecularly
characterize apparently balanced
rearrangements, and may find that these
rearrangements are more often pathogenic
than previously appreciated
©2016 MFMER | slide-19
Take Home Points
• Apparently balanced rearrangements represent an
interpretive challenge for clinical laboratories
• Many apparently balanced rearrangements are
not causative of abnormal phenotypes
• Potential pathogenic outcomes occur due to
multiple mechanisms, including gene disruption
• Limited tools to assess breakpoints at a high
resolution
• Next generation mate pair sequencing is a
methodology that can be used to molecularly
characterize structural rearrangements for clinically
useful interpretation and counseling
©2016 MFMER | slide-20
The Team….
• Nicole Hoppman, PhD
• Sarah Johnson
• Umut Aypar, PhD
• George Vazmatsis
• Erik Thorland, PhD
• Clinical Cytogenetics
Laboratory
• Hutton Kearney, PhD
• Geoffrey Halling
• Clinical Genome
Sequencing
Laboratory
• Kathryn Pearce
• Biomarker Discovery
• Stephanie Smoley
©2016 MFMER | slide-21
Questions??
©2016 MFMER | slide-22
Mayo Clinic
Locations
©2016 MFMER | slide-23