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Diamond-Blackfan Anemia Gene Discovery

Diamond-Blackfan Anemia Gene Discovery. Hanna T. Gazda, M.D., Ph.D. Boston Children’s Hospital Harvard Medical School Boston, MA. Boston Children’s Hospital Boston, MA. Genetic DBA projects. DBA gene discovery Modifier genes. DBA gene discovery project.

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Diamond-Blackfan Anemia Gene Discovery

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  1. Diamond-Blackfan Anemia Gene Discovery Hanna T. Gazda, M.D., Ph.D. Boston Children’s Hospital Harvard Medical School Boston, MA

  2. BostonChildren’s Hospital Boston, MA

  3. Genetic DBA projects • DBA gene discovery • Modifier genes

  4. DBA gene discovery project • Boston Children’s Hospital (Genetics)– Hanna Gazda, Daniel Yuan, Shideh Kazerounian, Lindsay Swanson • Broad Institute, Cambridge, MA – Vijay Sankaran, Eric Lander

  5. Objectives of the presentation • Ribosomal protein genes mutated in DBA • GATA1 mutated in DBA • What does it mean for DBA families?

  6. 5S 5.8SrRNA 28S 47 RPL 33 RPS 18SrRNA Ribosomal components 60S RPS19 RPS24 40S

  7. Hypothesis – Other ribosomal protein (RP) gene mutations may also cause DBA Aim of the Study – To screen remaining 78 ribosomal protein genes for mutations in DBA patients without known RPS19 and RPS24 mutations

  8. Methods – Screened DNA samples from DBA patients by direct sequencing of exons and intron/exon boundaries usingDNA from 96 DBA patients Sequence change identified • Sequencing of DNA from an additional 96 patients • Search the NCBI and HapMap SNP databases • Sequencing of DNA from 150-200 control samples • Sequencing of DNA from family members

  9. Analysis of sequencing data

  10. Analysis of sequencing data

  11. Summary of ribosomal protein genes mutated in DBA Draptchinskaia et al 1999 Gazda et al 2006 Cmejla et al 2007 Farrar et al 2008 Gazda et al 2008 Doherty et al 2010 Gazda et al 2012

  12. Large RP gene deletions in DBA • Dr. Bodine’s group (NIH); 9/51 • RPS19, RPS17, RPS26 and RPL35A • Dr. Hamaguchi’s group (Japan); 7/27 • RPS19, RPS17, RPL5 and RPL35A • Dr. Dianzani-Ramenghi’s group (Italy); 14/72 • RPS19, RPS17, RPS26, RPL5, RPL11 and RPL35A • Our own data (BCH); 6/87 • RPS19, RPS17, RPS24, RPS26 and RPL15

  13. Ribosomal protein genes and DBA • Ribosomal protein gene mutations and large deletions are known in about 60-65% of DBA patients • ~35-40% of patients do not have known pathogenic mutation(s)

  14. Importance of genetic screening in DBA • To confirm the clinical diagnosis of DBA • For stem cell transplantation • For reproductive choices (pre-implantation genetic diagnosis) • For future gene therapy

  15. RPL5mutation infamily with Aase syndrome wt wt Anemia Triphalangeal thumb VSD Anemia Triphalangeal thumb Cleft lip Aase JM & Smith DW, 1969 wt wt II-1, II-3, III-3 Exon5 indel wt wt

  16. Malformations in patients with RPL5, RPL11 and RPS19 mutations *Willig T-N et al, 1999; Rumenghi et al, 2000; Cmejla et al, 2000; Orfali et al, 2004 • Cleft lip/cleft palate RPL5 vs RPL11p=0.007; RPL5 vs RPS19p=9.745x10-7 • Thumb abnormalities RPL5 vs RPS19p=0.0024; RPL11 vs RPS19p=0.0012 • Congenital heart defects RPL5 vs RPS19p=0.017 • Multiple abnormalities RPL5 vs RPL11p=0.02; RPL5 vs RPS19p=0.0047

  17. Mutations of ribosomal protein genes in DBA Mutations ofRPS19, RPL5, RPL11, RPS10,RPS26 and RPL35Aare common causes of Diamond-Blackfan anemia, while RPS24, RPS7, RPS17, and RPL26 are sporadically mutated in DBA. All mutations are heterozygous and present in ~55% of patients. Mutations in RPL5 are associated with multiple physical abnormalities including triphalangeal thumbs and cleft lip/cleft palate, while RPL11 mutations are predominantly associated with isolated abnormal thumbs Large deletions are present in ~ 5-10% of patients. RPL15 is a novel gene associated with DBA.

  18. Mutations of ribosomal protein genes in DBA • Majority are nonsense, splice site or frameshift (insertions, deletions) • Heterozygous (present on one copy of the gene) and indicate autosomal dominant inheritance

  19. Karyogram of a human female

  20. Autosomal dominant inheritance http://www.uic.edu/nursing/genetics/Lecture/Types/SingleGene/AutosomalDominant/AD.htm

  21. Recurrencerisk of DBA Recurrence risk = 50%

  22. Reduced penetrance and variable expressivity in DBA I-1 I-2 M II-1 II-2 II-3 III-1 III-2 eADA eADA Normal eADA MCV MCV Normal MCV

  23. Variable expressivity in DBA I-1 I-2 eADA MCV n HbF II-1 II-2 II-5 II-3 II-4 II-6 II-7 n eADA n eADA n eADA n eADA n eADA eADA n MCV MCV n MCV n MCV n MCV n MCV n HbF n HbF n HbF n HbF n HbF n HbF III-1 III-2 III-3 eADA n eADA eADA n MCV  MCV  MCV n HbF  HbF  HbF

  24. Germline mutations in DBA N eADA N eADA N MCV N MCV M M  eADA  eADA  MCV  MCV

  25. Recurrencerisk of DBA I-1 I-2 I-1 I-2 M M II-1 II-2 II-3 II-1 II-2 II-3 III-1 III-2 III-1 III-2 ? ? ? ?

  26. Recurrencerisk of DBA Recurrence risk is slightly higher than in general population

  27. Ribosomal protein genes and DBA • Ribosomal protein gene mutations and large deletions are known in about 60-65% of DBA patients • ~35-40% of patients do not have known pathogenic mutation(s)

  28. Next step in DBA gene discoveries • Entire exome sequencing- (all exons) all coding regions of ~25,000 genes

  29. New patients enrolled into our study • 11 ribosomal protein gene screening • GATA1 gene screening • Screening of the new genes by exome sequencing

  30. Study participation inDBA gene discovery and modifier genes • Consent form and Questionnaire – Lindsay Swanson, genetic counselor; ph. 617-919-2169; lindsay.swanson@childrens.harvard.edu • Blood draw at local doctor’s office • Blood sample sent to Boston Children’s Hospital • No charge to participate

  31. Acknowledgements ] Alan H. Beggs Mee Rie Sheen Natasha Darras Leana Doherty Mike Landowski Chris Buros Roxy Ghazvinian Adrianna Vlachos Jeffrey M. Lipton Eva Atsidaftos Colin A. Sieff Sarah E. Ball Edyta Niewiadomska Michal Matysiak Peter E. Newburger ] Broad Institute, Cambridge, MA Genetics/Genomics Children’s Hospital Harvard Medical School Boston, MA, USA Vijay Sankaran Eric Lander ] Stanford University School of Medicine Stanford, CA Bertil Glader ] DBA Registry Feinstein Institute for Medical Research, Manhasset, NY ] University of Freiburg, Freiburg, Germany Charlotte Niemeyer Joerg Meerpohl Children’s Hospital Boston, MA, USA St.George's University of London, London, UK ] DBA Foundation DMA Foundation University Medical School of Warsaw, Warsaw, Poland University of Massachusetts Medical School, Worcester, MA, USA We thank the physicians, DBA patients and their family members for participating in the study!

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