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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