Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Wigard P. Kloosterman

1,435 views

Published on

Characteristics & molecular effects of complex genomic rearrangements

  • Be the first to comment

  • Be the first to like this

Wigard P. Kloosterman

  1. 1. Detection of copy number variation and structural variation by mate-pair sequencing Wigard Kloosterman UMC Utrecht CPHx 13-06-2012
  2. 2. SOLiD mate-pair sequencing F R Genomic DNA molecule
  3. 3. 1-2-3-SV: population-based mate-pair clustering Csfasta (F3) Qual (F3) Csfasta (R3) Qual (R3) SAP42 Step 1 Size distributions BAM (F3) Potential SV BAM (R3) regions Multiple libraries Step 2 Step 3 Visualization SVs Verification assays Victor Guryev
  4. 4. Thousands of structural variant calls per sample
  5. 5. Population based filtering of SVs Family- or population-based filtering to detect unique structural variants Calling of variants without relatives
  6. 6. High validation rate
  7. 7. Mate-pair analysis of a complex rearrangement• Identification of de novo structural events which may be causal to congenital defects in child (multiple congenital abnormalities and mental retardation)• Can we confirm classical diagnosis (karyotyping)?• Can we find additional de novo events? Son 46,XY,t(1;10;4)(p32.4;q21.1;q23) Father Mother flat aCGH profile
  8. 8. Detection of de novo variants 1e+06 father 8e+05 6e+05 Number of clones father 144 mother 106 148 4e+05 2e+05 3230 0e+00 0 2000 4000 6000 8000 10000 Mate pair size 451 242 mother 2000000 1500000 Number of clones 25 1000000 child 500000 0 0 2000 4000 6000 8000 10000 CFM Mate pair size 2000000 child 1500000 Number of clones 1000000 500000 0 0 2000 4000 6000 8000 10000 Mate pair size
  9. 9. Double-stranded DNA breaks result in 12 de novo breakpoint-junctions 57519917 57521100 55793180 57521088 57523787 55793182 chr10 57519913 57523805 55792170 57524597 50761470 chr1 50761463102287386 105745953 102287791 102287798 chr4 104738996 105025700 104738136 105036708 105028395105035150 105745783 105745828 105029770 105036735 105028400 =double-stranded DNA breaks • Junctions form pairs on the reference genome (double-stranded breaks) Kloosterman et al, HMG, 2011
  10. 10. Reconstruction of rearranged chromosomesMate-pair:Karyotype:
  11. 11. Double-strand break signature
  12. 12. Chromothripsis • Chromothripsis: chromosome shattering • Proposed as a model to explain massive chromosome rearrangements in cancer • Seen in 2%-3% of human cancers • Non-replicative modelStephens et al Cell 2011
  13. 13. Characterizing constitutional chromothripsis• Selection of 10 complex rearrangements in patients with congenital defects• Inclusion based on > 2 breakpoints• 130 breakpoint junctions• 8 rearrangements show signs of chromosome shattering by double-strand breaks Kloosterman et al, Cell Reports, 2012
  14. 14. Copy neutral chromothripsis 23 breakpoint junctions involving 5 chromosomes in mother no losses observed by array analysis
  15. 15. Derivative chromosomesChild inherited 2 out of 5 derivative chromosomes (16 out 23 breakpoints) ……
  16. 16. Complex CNVs in child… leading to severe developmental abnormalities and mental retardation
  17. 17. Unbalanced chromothripsisLost fragments:
  18. 18. Predicted deletions confirmed by depth of coverage analysisCopy number 3 2 1 Deleted fragments
  19. 19. Breakpoint Characteristics• Similar characteristics as seen for NHEJ repair of chromosome breaks
  20. 20. Paternal origin of chromothripsis• Multiple deletion intervals in four patient all hint at a paternal origin of chromothripsis
  21. 21. Conclusions-Structural variation can be efficiently assayed using SOLiD mate-pairsequencing • Family- or population-based filtering for identification of unique (pathogenic) variants-Chromothripsis is a common driver of complex rearrangementsinvolved in developmental disorders • Occurs both in cancer and in patient with developmental defects-What are the functional consequences of individual rearrangements?Just identifying them is not enough! • Requires multi-level analysis at a systematic scale
  22. 22. The Cuppen Group Ron Hochstenbach Wijnand Roessingh Ellen van Binsbergen Ivo Renkens Marloes Hoogstraat Mark van RoosmalenJose van de Belt Stef van Lieshout Marco Koudijs Masoumeh Tavakoli Joost Vermaat Ies Nijman Martin Poot Slavik Koval Karen Duran Ewart de BruijnVictor Guryev Edwin Cuppen Emile Voest
  23. 23. AcknowledgementsUMC Utrecht, Netherlands• Mark van Roosmalen Hubrecht Institute, Netherlands• Masoumeh Tavakoli • Victor Guryev• Marco Koudijs • Sebastiaan van Heesch• Oscar Paling • Nico Lansu• Marlous Hoogstraat • Ewart de Bruijn• Mirjam de Pagter • Marieke Simonis• Karen Duran• Myrthe Jager University of Leiden, Netherlands• Ivo Renkens • Kerstin Hansson• Elly Ippel • Claudia Ruivenkamp• Ellen van Binsbergen • Arie van Haeringen• Ron Hochstenbach• Martin Poot Ghent University Hospital, Belgium• Koen Braat • Sarah Vergult• Paul Coffer • Björn Menten• Emile Voest Universitatsklinikum Essen, Germany• Edwin Cuppen • Eberhard PassargeUniversity of Milan, Italy Institute of Human Genetics,• Lucia Ballarati Würzburg, Germany• Daniela Giardino • Thomas Haaf• Lidia Larizza

×